An extracellular carboxylesterase from the basidiomycete Pleurotus sapidus hydrolyses xanthophyll esters

An extracellular enzyme capable of efficient hydrolysis of xanthophyll esters was purified from culture supernatants of the basidiomycete Pleurotus sapidus. Under native conditions, the enzyme exhibited a molecular mass of 430 kDa, and SDS-PAGE data suggested a composition of eight identical subunits. Biochemical characterisation of the purified protein showed an isoelectric point of 4.5, and ideal hydrolysis conditions were observed at pH 5.8 and 40 degrees C. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. An 1861-bp cDNA containing an open reading frame of 1641 bp was cloned from a cDNA library that showed ca. 40% homology to Candida rugosa lipases. The P. sapidus carboxylesterase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus that has been characterised at the molecular level.     

Superior activity of the type C class of ISS in vitro and in vivo across multiple species

CpG-C are a novel class of CpG motif-containing immunostimulatory sequences (ISS) that includes both a 5'-TCG element and a CpG-containing palindrome. CpG-C drive all known ISS activities and, in particular, are potent enhancers of IFN-alpha from plasmacytoid dendritic cells (PDCs). In our examination of CpG-C sequence requirements, we determined that optimal IFN-alpha-inducing activity could be achieved with longer palindromes. Longer palindromes also correlated with maintenance of the double-stranded (ds) form despite concentration and pH changes, indicating a preference for ds oligodeoxynucleotides (ODNs) by the ISS-induced signaling mechanism for IFN-alpha synthesis. This correlation did not hold for all arms of the ISS-induced immune response, since we did not observe increased B cell activity with the longer palindrome CpG-C ODNs. We further demonstrated that CpG-C retained activity in an in vitro primate system and induced the expression of several cytokines and IFN-alpha-inducible genes when CpG-C were administered in vivo to mice and primates. In conclusion, we have shown CpG-C to exert several types of immune functions across multiple species, and this novel class is thus an attractive candidate for ISS-based therapeutic strategies.     

Identification of a positively evolving putative binding region with increased variability in posttranslational motifs in zonadhesin MAM domain 2

Positive selection has been shown to be pervasive in sex-related proteins of many metazoan taxa. However, we are only beginning to understand molecular evolutionary processes on the lineage to humans. To elucidate the evolution of proteins involved in human reproduction, we studied the sequence evolution of MAM domains of the sperm-ligand zonadhesin in respect to single amino acid sites, solvent accessibility, and posttranslational modification. GenBank-data were supplemented by new cDNA-sequences of a representative non-human primate panel. Solvent accessibility predictions identified a probably exposed fragment of 30 amino acids belonging to MAM domain 2 (i.e., MAM domain 3 in mouse). The fragment is characterized by significantly increased rate of positively selected amino acid sites and exhibits high variability in predicted posttranslational modification, and, thus, might represent a binding region in the mature protein. At the same time, there is a significant coincidence of positively selected amino acid sites and non-conserved posttranslational motifs. We conclude that the binding specificity of zonadhesin MAM domains, especially of the presumed epitope, is achieved by positive selection at the level of single amino acid sites and posttranslational modifications, respectively.     

Comparison of microsatellite and single nucleotide polymorphism markers for the genetic analysis of a Galloway cattle population

Highly informative genetic markers are essential for efficient management of cattle populations, as well as for food safety. After a decade of domination by microsatellite markers, a new type of genetic marker, single nucleotide polymorphism (SNP), has recently appeared on the scene. In the present study, the exclusion power of both kinds of markers with regards to individual identification and parental analysis was directly compared in a Galloway cattle population. Seventeen bovine microsatellites were distributed in three incremental marker sets (10, 14 and 17 microsatellite markers) and used for cattle genotyping. A set of 43 bovine SNP was used for genotyping the same cattle population. The accuracy of both kinds of markers in individual identification was evaluated using probability of identity estimations. These were 2.4 x 10(-8) for the 10 microsatellite set, 2.3 x 10(-11) for the 14 microsatellite set, and 1.4 x 10(-13) for the 17 microsatellite marker set. For the 43 SNP markers, the estimated probability of identity was 5.3 x 10(-11). The exclusion power of both kinds of markers in parental analysis was evaluated using paternity exclusion estimations, and, in addition to this, by estimation of the parental exclusion probability in 18 Galloway family trios. Paternity exclusion was estimated to be over 99% for microsatellites, and approx. 98% for SNP. Both, microsatellite and SNP sets of markers showed similar parental exclusion probabilities.     

Tumor promoter TPA stimulates MMP-9 secretion from human keratinocytes by activation of superoxide-producing NADPH oxidase

Matrix metalloproteinase-9 (MMP-9) is involved in physiological tissue remodelling processes as well as in tumor invasion and metastasis. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases MMP-9 secretion from normal human epidermal keratinocytes (NHEK) in vivo and in vitro. Here we show that the flavoprotein inhibitor diphenyleneiodinium (DPI) and the NADPH oxidase inhibitor apocynin block TPA-induced MMP-9 secretion of NHEK in vitro. Furthermore, N-acetyl-L-cysteine and L-cysteine lowered TPA-induced MMP-9 secretion, suggesting an involvement of reactive oxygen species(ROS). TPA exerts its effect on MMP-9 gene expression and secretion via the superoxide-producing enzyme NADPH oxidase: TPA rapidly stimulates generation of superoxide anion as well as gene expression of two cytosolic NADPH oxidase subunits (p47-phox and p67-phox) after 2 h, which is followed by induction of MMP-9 gene expression after 4 h. Taken together, the novel finding herein is the TPA-induced MMP-9 secretion from normal human epidermal keratinocytes through a NADPH oxidase dependent pathway.     

Dysregulation of bacterial proteolytic machinery by a new class of antibiotics

Here we show that a new class of antibiotics-acyldepsipeptides-has antibacterial activity against Gram-positive bacteria in vitro and in several rodent models of bacterial infection. The acyldepsipeptides are active against isolates that are resistant to antibiotics in clinical application, implying a new target, which we identify as ClpP, the core unit of a major bacterial protease complex. ClpP is usually tightly regulated and strictly requires a member of the family of Clp-ATPases and often further accessory proteins for proteolytic activation. Binding of acyldepsipeptides to ClpP eliminates these safeguards. The acyldepsipeptide-activated ClpP core is capable of proteolytic degradation in the absence of the regulatory Clp-ATPases. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death.     

Modification of the structure and function of fibrillin-1 by homocysteine suggests a potential pathogenetic mechanism in homocystinuria

Homocystinuria, a disorder originating in defects in the methionine metabolism, is characterized by an elevated plasma concentration of homocysteine. Most patients have a defect in the cystathionine-beta-synthase, the key enzyme in the conversion of homocysteine to cysteine. Many abnormalities in the connective tissue of patients with homocystinuria resemble those seen in Marfan syndrome, caused by mutations in fibrillin-1. These observations led to the hypothesis that the structure and function of fibrillin-1 is compromised in patients with homocystinuria. To test this hypothesis we produced recombinant human fibrillin-1 fragments spanning the central portion of the molecule (8-Cys/transforming growth factor-beta binding domain 3 to calcium binding EGF domain 22) and extensively analyzed the potential of homocysteine to modify structural and functional properties of these proteins. Circular dichroism spectroscopy revealed moderate changes of their secondary structures after incubation with homocysteine. Equilibrium dialysis demonstrated a number of high affinity calcium binding sites in the tandemly repeated calcium binding epidermal growth factor-like domains 11-22. Calcium binding of homocysteine-modified fragments was completely abolished. Incubation of the recombinant proteins with homocysteine rendered the analyzed calcium binding EGF domains as well as the 8-Cys/transforming growth factor-beta binding domain 3 significantly more susceptible to proteolytic degradation. Furthermore, data were obtained demonstrating that homocysteine can covalently modify fibrillin-1 via disulfide bonds. These data strongly suggest that structural and functional modifications as well as degradation processes of fibrillin-1 in the connective tissues of patients with homocystinuria play a major role in the pathogenesis of this disorder.     

Site-directed mutagenesis of the active site region in the quinate/shikimate 5-dehydrogenase YdiB of Escherichia coli

YdiB and its paralog AroE are members of the quinate/shikimate 5-dehdrogenase family. Enzymes from this family function in the shikimate pathway that is essential for survival of microorganisms and plants and represent potential drug targets. Recent YdiB and AroE crystal structures revealed the presence of a NAD(P)-binding and a catalytic domain. We carried out site-directed mutagenesis of 8 putative active site residues in YdiB from Escherichia coli and analyzed structural and kinetic properties of the mutant enzymes. Our data indicate critical roles for an invariant lysine and aspartate residue in substrate binding and allowed us to differentiate between two previously proposed models for the binding of the substrate in the active site. Comparison of several YdiB and AroE structures led us to conclude that, upon cofactor binding and domain closure, the 2 identified binding residues are repositioned to bind to the substrate. Although the lysine residue contributes to some extent to the stabilization of the transition state, we did not identify any residue as catalytically essential. This indicates that catalysis does not operate through a general acid-base mechanism, as thought originally. Our improved understanding of the medically and agriculturally important quinate/shikimate 5-dehydrogenase family at the molecular level may prove useful in the development of novel herbicides and antimicrobial agents.