Crystal structures of SnoaL2 and AclR: two putative hydroxylases in the biosynthesis of aromatic polyketide antibiotics

SnoaL2 and AclR are homologous enzymes in the biosynthesis of the aromatic polyketides nogalamycin in Streptomyces nogalater and cinerubin in Streptomyces galilaeus, respectively. Evidence obtained from gene transfer experiments suggested that SnoaL2 catalyzes the hydroxylation of the C-1 carbon atom of the polyketide chain. Here we show that AclR is also involved in the production of 1-hydroxylated anthracyclines in vivo. The three-dimensional structure of SnoaL2 has been determined by multi-wavelength anomalous diffraction to 2.5A resolution, and that of AclR to 1.8A resolution using molecular replacement. Both enzymes are dimers in solution and in the crystal. The fold of the enzyme subunits consists of an alpha+beta barrel. The dimer interface is formed by packing of the beta-sheets from the two subunits against each other. In the interior of the alpha+beta barrel a hydrophobic cavity is formed that most likely binds the substrate and harbors the active site. The subunit fold and the architecture of the active site in SnoaL2 and AclR are similar to that of the polyketide cyclases SnoaL and AknH; however, they show completely different quaternary structures. A comparison of the active site pockets of the putative hydroxylases AclR and SnoaL2 with those of bona fide polyketide cyclases reveals distinct differences in amino acids lining the cavity that might be responsible for the switch in chemistry. The moderate degree of sequence similarity and the preservation of the three-dimensional fold of the polypeptide chain suggest that these enzymes are evolutionary related. Members of this enzyme family appear to have evolved from a common protein scaffold by divergent evolution to catalyze reactions chemically as diverse as aldol condensation and hydroxylation.     

Cloning, characterization and localization of three novel class III peroxidases in lignifying xylem of Norway spruce (Picea abies)

Plant class III peroxidases (POXs) take part in the formation of lignin and maturation of plant cell walls. However, only a few examples of such peroxidases from gymnosperm tree species with highly lignified xylem tracheids have been implicated so far. We report here cDNA cloning of three xylem-expressed class III peroxidase encoding genes from Norway spruce (Picea abies). The translated proteins, PX1, PX2 and PX3, contain the conserved amino acids required for heme-binding and peroxidase catalysis. They all begin with putative secretion signal propeptide sequences but diverge substantially at phylogenetic level, grouping to two subclusters when aligned with other class III plant peroxidases. In situ hybridization analysis on expression of the three POXs in Norway spruce seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm of young, developing tracheids within the current growth ring where lignification is occurring. Function of the putative N-terminal secretion signal peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions with EGFP (enhanced green fluorescent protein) and expressing them in tobacco protoplasts. Full-length coding region of px1 was also heterologously expressed in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1 peroxidase is processed via the endoplasmic reticulum (ER) most likely for secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal localization for participation in the maturation of the spruce tracheid secondary cell wall.     

Cardiac dysfunction in transgenic mouse fetuses overexpressing shortened type XIII collagen

Overexpression of type XIII collagen molecules with an 83-amino-acid residue in-frame deletion of part of the ectodomain leads to fetal lethality in Col13a1 (COL2del) transgenic mice. We characterize here the functional disturbances in the cardiovascular system of mouse fetuses overexpressing mutant type XIII collagen. Doppler ultrasonography was performed at 12.5 days of gestation on 33 fetuses resulting from heterozygous matings of seven female mice and on 16 fetuses from two matings between heterozygous and wild-type mice. Nine fetuses had atrioventricular valve regurgitation (AVVR), and all of them were transgene-positive. The fetuses with AVVR had a lower outflow mean velocity (V(mean); P < 0.005) and a greater proportion of isovolumetric relaxation time (IRT%) in the cardiac cycle (P < 0.0001) than those without AVVR, and their ductus venosus pulsatility indices for veins (DV PIV) and the umbilical artery pulsatility indices were increased. A positive correlation was found between IRT% and DV PIV, and a negative correlation was seen between outflow V(mean) and DV PIV. Morphological analysis of the heart revealed no differences between the two groups of fetuses, but histological analysis showed the trabeculation of the ventricles to be reduced and the myocardium to be thinner in the fetuses with AVVR. Based on in situ hybridization, type XIII collagen mRNAs were normal constituents of these structures. Moreover, a positive correlation was found between outflow V(mean) and myocardial thickness. IRT% and DV PIV correlated negatively with myocardial thickness. Thus, overexpression of mutant type XIII collagen results in mid-gestation cardiac dysfunction in mouse fetuses, and these disturbances in cardiac function may lead to death in utero.     

Lack of collagen XVIII accelerates cutaneous wound healing, while overexpression of its endostatin domain leads to delayed healing

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII (Col18a1(-/-)). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1(-/-) mice, and the vascularisation rate was accelerated in the Col18a1(-/-) mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half (n=10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal-dermal junction in the presence of an excess of endostatin.     

A Cryptic Frizzled Module in Cell Surface Collagen 18 Inhibits Wnt/β?Catenin Signaling

Collagens contain cryptic polypeptide modules that regulate major cell functions, such as cell proliferation or death. Collagen XVIII (C18) exists as three amino terminal end variants with specific amino terminal polypeptide modules. We investigated the function of the variant 3 of C18 (V3C18) containing a frizzled module (FZC18), which carries structural identity with the extracellular cysteine-rich domain of the frizzled receptors. We show that V3C18 is a cell surface heparan sulfate proteoglycan, its topology being mediated by the FZC18 module. V3C18 mRNA was expressed at low levels in 21 normal adult human tissues. Its expression was up-regulated in fibrogenesis and in small well-differentiated liver tumors, but decreased in advanced human liver cancers. Low FZC18 immunostaining in liver cancer nodules correlated with markers of high Wnt/β−catenin activity. V3C18 (M_r = 170 kD) was proteolytically processed into a cell surface FZC18-containing 50 kD glycoprotein precursor that bound Wnt3a in vitro through FZC18 and suppressed Wnt3a-induced stabilization of β−catenin. Ectopic expression of either FZC18 (35 kD) or its 50 kD precursor inhibited Wnt/β−catenin signaling in colorectal and liver cancer cell lines, thus downregulating major cell cycle checkpoint gatekeepers cyclin D1 and c-myc and reducing tumor cell growth. By contrast, full-length V3C18 was unable to inhibit Wnt signaling. In summary, we identified a cell-surface signaling pathway whereby FZC18 inhibits Wnt/β−catenin signaling. The signal, encrypted within cell-surface C18, is released by enzymatic processing as an active frizzled cysteine-rich domain (CRD) that reduces cancer cell growth. Thus, extracellular matrix controls Wnt signaling through a collagen-embedded CRD behaving as a cell-surface sensor of proteolysis, conveying feedback cues to control cancer cell fate.     

Proteolytic Function of GPI-Anchored Plasma Membrane Protease Yps1p in the Yeast Vacuole and Golgi

Yps1p is a member of the GPI-anchored aspartic proteases which reside at the plasma membrane of Saccharomyces cerevisiae. Here we show that in Δerg6 cells, where a late biosynthetic step of the membrane lipid ergosterol is blocked, part of Yps1p was targeted to the vacuole. There it overtook proteolytic functions of the Pep4p protease, resulting in processing of pro-CPY to CPY in cells lacking the PEP4 gene. Yps1p was enriched in membrane microdomains, as it could be isolated in detergent-insoluble complexes from both normal and Δerg6 cells. Vacuolar Yps1 caused degradation of a mammalian sialyltransferase ectodomain fusion protein (ST6Ne), which was directed from the Golgi to the vacuole in both normal and Δerg6 cells. Unexpectedly, ST6Ne was degraded also when arrested in the Golgi in a temperature-sensitive sec7–1 mutant. Newly synthesized Yps1p, in transit to the plasma membrane, was also involved in the Golgi-associated degradation. These data show that GPI-anchored proteases, whose biological roles are unknown, may reside and function in different subcellular locations.     

Thermoregulatory responses of two mouse Mus musculus strains selectively bred for high and low food intake

We examined the thermoregulatory responses of male and female mice Mus musculus that had been divergently selected on voluntary food intake, corrected for body mass, to produce a high-intake and a low-intake strain. Resting metabolic rate was determined by indirect calorimetry (at 30 degrees C, 25 degrees C, 15 degrees C and 5 degrees C). Body temperature responses were measured in a separate group of mice in a parallel protocol. High-intake mice had significantly elevated body masses compared to low-intake mice in both sexes. Lower critical temperature in both strains appeared to be around 28 degrees C. At 30 degrees C there was a significant strain effect on resting metabolic rate, with high strain mice having greater metabolism than low strain mice. Sex and body mass were not significant main effects on resting metabolic rate and there were no significant interactions. Body temperature measured at 30 degrees C, 25 degrees C, 15 degrees C and 5 degrees C differed significantly between sexes (females higher) and there was a significant sexxbody mass interaction effect, but there was no difference between strains. Thermal conductance was significantly related to strain and sex, mice from the high strain and males having greater thermal conductances than mice from the low strain and females. Artificial selection has resulted in high-intake mice having greater body masses and greater thermal conductances, which together account for up to 45% of the elevated daily energy demands that underpin the increase in food intake. The greater levels of food intake were also associated with higher resting metabolic rates at 30 degrees C.     

Conversion of Milled Pine Wood by Manganese Peroxidase from Phlebia radiata

Purified manganese peroxidase (MnP) from the white-rot basidiomycete Phlebia radiata was found to convert in vitro milled pine wood (MPW) suspended in an aqueous reaction solution containing Tween 20, Mn²⁺, Mn-chelating organic acid (malonate), and a hydrogen peroxide-generating system (glucose-glucose oxidase). The enzymatic attack resulted in the polymerization of lower-molecular-mass, soluble wood components and in the partial depolymerization of the insoluble bulk of pine wood, as demonstrated by high-performance size exclusion chromatography (HPSEC). The surfactant Tween 80 containing unsaturated fatty acid redsidues promoted the disintegration of bulk MPW. HPSEC showed that the depolymerization yielded preferentially lignocellulose fragments with a predominant molecular mass of ca. 0.5 kDa. MnP from P. radiata (MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37°C with vigorous stirring, and 65 and 35% of the activity applied was retained in Tween 20 and Tween 80 reaction mixtures, respectively. In the course of reactions, major part of the Mn-chelator malonate was decomposed (85 to 87%), resulting in an increase of pH from 4.4 to >6.5. An aromatic nonphenolic lignin structure (β-O-4 dimer), which is normally not attacked by MnP, was oxidizible in the presence of pine wood meal. This finding indicates that certain wood components may promote the degradative activities of MnP in a way similar to that promoted by Tween 80, unsaturated fatty acids, or thiols.