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.

The PcACE1 transcription factor from Phanerochaete chrysosporium contains a Cys- and Ser-rich transactivation domain.

Transcription factor Ace1 from Saccharomyces cerevisiae regulates the expression of target genes when the copper concentration reaches 200 ìÌ levels. We are studying the ortholog of Ace1 from fungus Phanerochaete chrysosporium PcACE1, isolated by complementation in yeast. In this report we show the localization of the transactivation region of PcACE1. Different PcACE1 fragments were ligated in frame to the GAL4 DNA-binding domain by site-directed mutagenesis in a suitable yeast expression vector. Transformation of an appropriate Saccharomyces cerevisiae strain was used as host. This strain contains the fusion GAL1:lacZ in its genome under the control of promoter sequences recognized by GAL4. Finally, we measured â-galactosidase activity in each yeast clone. The activation of the reporter gene is proportional to the transactivation capacity of the transcription factor PcACE1. The results obtained indicate that PcACE1 transactivation domain is located in the carboxy terminal half and contains an array of cysteines in the form of Cys-X-Cys and Cys-X2-Cys and a 60% of Ser. Therefore, these results show that this type of Cys motif can function as transcription activating domain not only in transcription factors that respond to minimal copper concentrations but also in those that respond to high copper concentrations. This is the first transactivation domain reported in a basidiomycete fungus.

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 P2X(2)R and inhibits the activity of P2X(4)R. 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 P2X(2) subunit, termed P2X(2a), and a splice variant lacking a 69 residue segment in the C terminal, termed P2X(2b), as the donors for intracellular and transmembrane segments and the P2X(4) subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X(4/2a) chimera but was absent in oocytes expressing P2X(4/2b) chimera. Site-directed mutagenesis experiments revealed that the Cys(430) residue mediates effects of mercury on the P2X(2a)R activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide (H(2)O(2)) 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 P2X(2a)R and P2X(4/2a)R currents but not P2X(2b)R and P2X(2a)-C430A and P2X(2a)-C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the H(2)O(2) potentiation. Alkylation of Cys(430) residue with methylmethane-thiosulfonate also abolished the mercury and H(2)O(2) potentiation. Altogether, these results are consistent with the hypothesis that the Cys(430) residue is an intracellular P2X(2a)R redox sensor.

The xylanolytic enzyme system from the genus Penicillium.

In nature, there are numerous microorganisms that efficiently degrade xylan, a major component of lignocellulose. In particular, filamentous fungi have demonstrated a great capability for secreting a wide range of xylanases, being the genus Aspergillus and Trichoderma the most extensively studied and reviewed among the xylan-producing fungi. However, an important amount of information about the production and genetics of xylanases from fungi of the genus Penicillium has accumulated in recent years. A great number of Penicillia are active producers of xylanolytic enzymes, and the use of xylanases from these species has acquired growing importance in biotechnological applications. This review summarizes our current knowledge about the properties, genetics, expression and biotechnological potential of xylanases from the genus Penicillium.

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.

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

1. 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-CH(3)=2C-D analogs, are partial agonists at 5-HT(2C) receptors, and show low or even negligible intrinsic efficacy at 5-HT(2A) receptors. These results raised the proposal that these drugs may act as 5-HT(2) antagonists. 2. To test this hypothesis, Xenopus laevis oocytes were microinjected with the rat clones for 5-HT(2A) or 5-HT(2C) receptors. The above-mentioned PEAs and its 4-H analog (2C-H) blocked the 5-HT-induced currents at 5-HT(2A), but not at the 5-HT(2C) receptor, revealing 5-HT(2) receptor subtype selectivity. The 5-HT(2A) receptor antagonism required a 2-min preincubation to attain maximum inhibition. 3. 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-HT(2A) receptor antagonist potency was 2C-I>2C-B>2C-D>2C-H. 4. The present results demonstrate that in X. laevis oocytes, a series of 2,5-dimethoxy-4-substituted PEAs blocked the 5-HT(2A) but not the 5-HT(2C) 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-HT(2A) receptor agonism.

Secretion of endoxylanase A from Penicillium purpurogenum by Saccharomyces cerevisiae transformed with genomic fungal DNA.

Saccharomyces cerevisiae was transformed with a genomic library from Penicillium purpurogenum, and an endoxylanase-producing yeast clone (named 44A) that grows on xylose or xylan as sole carbon source was isolated. This yeast synthesizes xynA mRNA and secretes endoxylanase A to culture media when grown on xylan or xylose, but not glucose. Analysis by pulse-field gel electrophoresis and sequencing indicates that xynA, including its eight introns, has been inserted into the yeast genome. It was shown by sequencing that clone 44A is able to correctly splice xynA introns. This is the first successful attempt to express a fungal endoxylanase gene in yeast with correct intron splicing.

Functional analysis of the endoxylanase B (xynB) promoter from Penicillium purpurogenum.

In Penicillium purpurogenum, the gene encoding endoxylanase B (xynB) is highly expressed by xylan and repressed by glucose at the transcriptional level. The promoter of this gene has a modular structure, with eight putative XlnR binding sites in tandem (XlnR module), and upstream from them, four putative CreA binding sites (CreA module). Promoter fragments containing different modules were inserted into a plasmid, pAN49-1, which contains a basal fungal promoter linked to a reporter gene (lacZ) and its expression was studied in vivo in Aspergillus nidulans. The XlnR module is able to trigger high beta-galactosidase activity in the presence of xylan, but the lack of most XlnR sites notoriously reduces this enzymatic activity. No enzyme induction is observed if the orientation of the promoter fragment is inverted. The presence of the CreA module is necessary for glucose repression when beta-galactosidase activity is previously induced by xylan. However, when transformant strains containing the XlnR module but lacking all CreA sites were grown in glucose without pre-induction in xylan, a low beta-galactosidase activity was observed compared with the same transformants grown in xylan. These results agree with a double-lock regulatory mechanism for both direct and indirect repression of xylanolytic genes by glucose.

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.

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.