Paulina Bull

Differences in expression of two endoxylanase genes (xynA and xynB) from Penicillium purpurogenum.

A number of xylanolytic microorganisms secrete to the medium several molecular forms of endoxylanases. The physiological function of these isoforms is not clear; one possibility is that they are produced under different growth conditions. To study this problem, we have used two endoxylanases (XynA and XynB) produced by the fungus Penicillium purpurogenum. These enzymes have been previously purified and characterized; they belong to family 10 and 11 of the glycosyl hydrolases, respectively. The promoters of the xynA and xynB genes have been sequenced; both present consensus sequences for the binding of the carbon catabolite repressor CreA, but otherwise show substantial differences. The xynB promoter has eight boxes in tandem for the binding of the XlnR activator and lacks the consensus sequence for the PacC pH regulator. On the other hand, the xynA promoter contains one XlnR box and three PacC consensus sequences. To investigate if these differences are reflected in gene expression, Northern blot assays were carried out. The xynA gene is transiently expressed when oat spelt xylan is used as carbon source, but negligible expression was observed with birchwood xylan, xylose or xylitol. In contrast, xynB is broadly induced by all these carbon sources; this may be related to the presence of several XlnR boxes. Similar results were obtained by zymogram analysis of the expressed proteins. The different induction capabilities of birchwood and oat spelt xylan may be due to differences in their composition and structure. Expression assays carried out at different pH reflects that, despite the lack of PacC binding sites in the xynB promoter, this gene is tightly regulated by pH. The findings described here illustrate new and important differences between endoxylanases from families 10 and 11 in P. purpurogenum. They may help explain the production of multiple endoxylanase forms by this organism.

Dissecting the Facilitator and Inhibitor Allosteric Metal Sites of the P2X4 Receptor Channel CRITICAL ROLES OF CYS132 FOR ZINC POTENTIATION AND ASP138 FOR COPPER INHIBITION

Zinc and copper are atypical modulators of ligand-gated ionic channels in the central nervous system. We sought to identify the amino acids of the rat P2X4 receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His140, a residue previously identified as being critical for copper-induced inhibition of the ATP-evoked currents. Site-directed mutagenesis replaced conspicuous amino acids located within the extracellular domain region between Thr123 and Thr146 for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys132, but not Cys126, proved crucial for zinc-induced potentiation of the receptor activity, but not for copper-induced inhibition. Zinc inhibited in a concentration-dependent manner the ATP-gated currents of the C132A mutant. Likewise, Asp138, but not Asp131 was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp129, Asp131, and Thr133 had minor roles in metal modulation. We conclude that this region of the P2X4 receptor has a pocket for trace metal coordination with two distinct and separate facilitator and inhibitor metal allosteric sites. In addition, Cys132 does not seem to participate exclusively as a structural receptor channel folding motif but plays a role as a ligand for zinc modulation highlighting the role of trace metals in neuronal excitability.

4-Bromo-2,5-dimethoxyphenethylamine (2C-B) and structurally related phenylethylamines are potent 5-HT2A receptor antagonists in Xenopus laevis oocytes

We recently described that several 2‐(2,5‐dimethoxy‐4‐substituted phenyl)ethylamines (PEAs), including 4‐I=2C‐I, 4‐Br=2C‐B, and 4‐CH3=2C‐D analogs, are partial agonists at 5‐HT2C receptors, and show low or even negligible intrinsic efficacy at 5‐HT2A receptors. These results raised the proposal that these drugs may act as 5‐HT2 antagonists. To test this hypothesis, Xenopus laevis oocytes were microinjected with the rat clones for 5‐HT2A or 5‐HT2C receptors. The above‐mentioned PEAs and its 4‐H analog (2C‐H) blocked the 5‐HT‐induced currents at 5‐HT2A, but not at the 5‐HT2C receptor, revealing 5‐HT2 receptor subtype selectivity. The 5‐HT2A receptor antagonism required a 2‐min preincubation to attain maximum inhibition. All PEAs tested shifted the 5‐HT concentration–response curves to the right and downward. Their potencies varied with the nature of the C(4) substituent; the relative rank order of their 5‐HT2A receptor antagonist potency was 2C‐I>2C‐B>2C‐D>2C‐H. The present results demonstrate that in X. laevis oocytes, a series of 2,5‐dimethoxy‐4‐substituted PEAs blocked the 5‐HT2A but not the 5‐HT2C receptor‐mediated responses. As an alternative hypothesis, we suggest that the psychostimulant activity of the PEAs may not be exclusively associated with partial or full 5‐HT2A receptor agonism.

Synaptic Clustering of PSD-95 Is Regulated by c-Abl through Tyrosine Phosphorylation

The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95.

Characterization of an α-L-arabinofuranosidase gene (abf1) from Penicillium purpurogenum and its expression

An alpha-L-arabinofuranosidase gene (abf1) from Penicillium purpurogenum was identified and sequenced. abf1 has an open reading frame of 1518 bp, does not contain introns and codes for a protein of 506 amino acids. The deduced mature protein has a molecular mass of 49.6 KDa, and its sequence is homologous to arabinofuranosidases of glycosyl hydrolase family 54. Southern blots suggest that abf1 is a single copy gene. Putative sequences for the binding of the transcriptional regulators XlnR, CreA, PacC, AlcR and AreA are present in the promoter. Northern-blot analysis shows that abf1 is expressed at neutral but not at alkaline or acidic pH values. The presence of binding sites for regulatory elements in the promoter region has been compared to the genes of other fungal enzymes belonging to the same family. This is the first characterization of an abf gene from a Penicillium species.

Inactivation of E. coli RNA polymerase by pyridoxal 5′-phosphate: Identification of a low pKa lysine as the modified residue

Abstract The inactivation of E. coli RNA polymerase (3.3 × 10 −7 M) by pyridoxal 5′-phosphate (1 × 10 −4 M to 5 × 10 −4 M) is a first order process with respect to the remaining active enzyme. Studies of the variation of the first order rate constant with the concentration of pyridoxal 5′-phosphate show that the inactivation reaction follows saturation kinetics. The formation of a reversible enzyme-inhibitor intermediate is postulated. Kinetic studies at different pH values indicate that the inactivation rate constant depends on the mole fraction of one conjugate base with pK a 7.9. The apparent equilibrium constant (association) for the inactivation reaction is independent of the pH and is 1.8 × 10 4 M −1 . By electrophoretic and chromatographic analysis of enzyme hydrolyzates after pyridoxal 5′-phosphate and NaBH 4 treatment only N-e-pyridoxyllysine was found. It is postulated that a lysine e-amino group with a low pK a is critical for the activity of the enzyme.

The half-life of the T-cell receptor/peptide–major histocompatibility complex interaction can modulate T-cell activation in response to bacterial challenge

T‐cell activation results from engagement of the T‐cell receptor (TCR) by cognate peptide–major histocompatibility complex (pMHC) complexes on the surface of antigen‐presenting cells (APC). Previous studies have provided evidence supporting the notion that the half‐life of the TCR/pMHC interaction and the density of pMHC on the APC are two parameters that can influence T‐cell activation. However, whether the half‐life of the TCR/pMHC interaction can modulate T‐cell activation in response to a pathogen challenge remains unknown. To approach this question, we generated strains of bacteria expressing variants of the ovalbumin (OVA) antigen, carrying point mutations in the SIINFEKL sequence. When bound to H‐2Kb, this peptide is the cognate ligand for the OT‐I TCR. Variants of the H‐2Kb/SIINFEKL bind to the OT‐I TCR with distinct half‐lives. Here we show that dendritic cells (DCs) infected with bacteria expressing OVA variants were incapable of activating OT‐I T cells when the half‐life of the TCR/H‐2Kb/OVA interaction was excessively short. Consistent with these data, T‐cell activation was only observed in mice infected with bacteria expressing OVA variants that bound to OT‐I with a half‐life above a certain threshold. Considered together, our data suggest that the half‐life of TCR/pMHC interaction can significantly modulate T‐cell activation in vivo, as well as influence recognition of antigens expressed by bacteria. These observations underscore the importance of the TCR/pMHC half‐life on the clearance of pathogens.

Reactive Oxygen Species Potentiate the P2X2 Receptor Activity through Intracellular Cys430

P2X receptor channels (P2XRs) are allosterically modulated by several compounds, mainly acting at the ectodomain of the receptor. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X2R and inhibits the activity of P2X4R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X2 subunit, termed P2X2a, and a splice variant lacking a 69 residue segment in the C terminal, termed P2X2b, as the donors for intracellular and transmembrane segments and the P2X4 subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X4/2a chimera but was absent in oocytes expressing P2X4/2b chimera. Site-directed mutagenesis experiments revealed that the Cys430 residue mediates effects of mercury on the P2X2aR activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide (H2O2) and mitochondrial stress inducers myxothiazol and rotenone mimicked mercury effects. These experiments, done in both oocytes and human embryonic kidney HEK293 cells, revealed that these compounds potentiated the ATP-evoked P2X2aR and P2X4/2aR currents but not P2X2bR and P2X2a–C430A and P2X2a–C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the H2O2 potentiation. Alkylation of Cys430 residue with methylmethane-thiosulfonate also abolished the mercury and H2O2 potentiation. Altogether, these results are consistent with the hypothesis that the Cys430 residue is an intracellular P2X2aR redox sensor.

The copper-dependent ACE1 transcription factor activates the transcription of the mco1 gene from the basidiomycete Phanerochaete chrysosporium.

We have previously identified and functionally characterized the transcription factor ACE1 (Pc-ACE1) from Phanerochaete chrysosporium. In Saccharomyces cerevisiae, ACE1 activates the copper-dependent transcription of target genes through a DNA sequence element named ACE. However, the possible target gene(s) of Pc-ACE1 were unknown. An in silico search led to the identification of putative ACE elements in the promoter region of mco1, one of the four clustered genes encoding multicopper oxidases (MCOs) in P. chrysosporium. Since copper exerts an effect at the transcriptional level in MCOs from several organisms, in this work we analysed the effect of copper on transcript levels of the clustered MCO genes from P. chrysosporium, with the exception of the transcriptionally inactive mco3. Copper supplementation of cultures produced an increment in transcripts from genes mco1 and mco2, but not from mco4. Electrophoretic mobility-shift assays revealed that Pc-ACE1 binds specifically to a probe containing one of the putative ACE elements found in the promoter of mco1. In addition, using a cell-free transcription system, we showed that in the presence of cuprous ion, Pc-ACE1 induces activation of the promoter of mco1, but not that of mco2.

Structure analysis of the endoxylanase. A gene from penicillium purpurogenum

Penicillium purpurogenum produces several endoxylanases, two of which (XynA and XynB) have been purified and characterized. XynB has been sequenced, and it belongs to glycosyl hydrolase family 11. In this publication we report the structure of the xynA gene. The amino terminal sequence of the protein was determined and this allowed the design of oligonucleotides for use in polymerase chain reactions. Different polymerase chain reaction strategies were used to amplify and sequence the entire cDNA and the gene. The gene has an open reading frame of 1450 base pairs, including 8 introns with an average length of 56 base pairs each. Only one copy of this gene is present in the P. purpurogenum genome as shown by Southern blot. The gene encodes a protein of 329 residues (including the signal peptide), and the calculated molecular mass of the mature protein is 31,668 Da. Immunodetection assays of the expressed gene positively identified it as xynA, and sequence alignments indicate a high degree of similarity with family 10 endoxylanases. It is concluded that P. purpurogenum produces endoxylanases of family 10 and 11. The complementary action of endoxylanases of both families may be important for an efficient degradation of xylan by the fungus.