Xenomic networks variability and adaptation traits in wood decaying fungi.

Fungal degradation of wood is mainly restricted to basidiomycetes, these organisms having developed complex oxidative and hydrolytic enzymatic systems. Besides these systems, wood-decaying fungi possess intracellular networks allowing them to deal with the myriad of potential toxic compounds resulting at least in part from wood degradation but also more generally from recalcitrant organic matter degradation. The members of the detoxification pathways constitute the xenome. Generally, they belong to multigenic families such as the cytochrome P450 monooxygenases and the glutathione transferases. Taking advantage of the recent release of numerous genomes of basidiomycetes, we show here that these multigenic families are extended and functionally related in wood-decaying fungi. Furthermore, we postulate that these rapidly evolving multigenic families could reflect the adaptation of these fungi to the diversity of their substrate and provide keys to understand their ecology. This is of particular importance for white biotechnology, this xenome being a putative target for improving degradation properties of these fungi in biomass valorization purposes.

The Escherichia coli SLC26 homologue YchM (DauA) is a C(4)-dicarboxylic acid transporter.

The SLC26/SulP (solute carrier/sulphate transporter) proteins are a ubiquitous superfamily of secondary anion transporters. Prior studies have focused almost exclusively on eukaryotic members and bacterial members are frequently classified as sulphate transporters based on their homology with SulP proteins from plants and fungi. In this study we have examined the function and physiological role of the Escherichia coli Slc26 homologue, YchM. We show that there is a clear YchM-dependent growth defect when succinate is used as the sole carbon source. Using an in vivo succinate transport assay, we show that YchM is the sole aerobic succinate transporter active at acidic pH. We demonstrate that YchM can also transport other C(4) -dicarboxylic acids and that its substrate specificity differs from the well-characterized succinate transporter, DctA. Accordingly ychM was re-designated dauA (dicarboxylic acid uptake system A). Finally, our data suggest that DauA is a protein with transport and regulation activities. This is the first report that a SLC26/SulP protein acts as a C(4) -dicarboxylic acid transporter and an unexpected new function for a prokaryotic member of this transporter family.

New substrates and activity of Phanerochaete chrysosporium Omega glutathione transferases.

Omega glutathione transferases (GSTO) constitute a family of proteins with variable distribution throughout living organisms. It is notably expanded in several fungi and particularly in the wood-degrading fungus Phanerochaete chrysosporium, raising questions concerning the function(s) and potential redundancy of these enzymes. Within the fungal families, GSTOs have been poorly studied and their functions remain rather sketchy. In this study, we have used fluorescent compounds as activity reporters to identify putative ligands. Experiments using 5-chloromethylfluorescein diacetate as a tool combined with mass analyses showed that GSTOs are able to cleave ester bonds. Using this property, we developed a specific activity-based profiling method for identifying ligands of PcGSTO3 and PcGSTO4. The results suggest that GSTOs could be involved in the catabolism of toxic compounds like tetralone derivatives. Biochemical investigations demonstrated that these enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteine residue. To access the physiological function of these enzymes and notably during the wood interaction, recombinant proteins have been immobilized on CNBr Sepharose and challenged with beech wood extracts. Coupled with GC-MS experiments this ligand fishing method allowed to identify terpenes as potential substrates of Omega GST suggesting a physiological role during the wood-fungus interactions.

Sphingobium sp. SYK-6 LigG involved in lignin degradation is structurally and biochemically related to the glutathione transferase ω class.

SpLigG is one of the three glutathione transferases (GSTs) involved in the process of lignin breakdown in the soil bacterium Sphingobium sp. SYK-6. Sequence comparisons showed that SpLigG and several proteobacteria homologues form an independent cluster within cysteine-containing GSTs. The relationship between SpLigG and other GSTs was investigated. The X-ray structure and biochemical properties of SpLigG indicate that this enzyme belongs to the omega class of glutathione transferases. However, the hydrophilic substrate binding site of SpLigG, together with its known ability to stereoselectively deglutathionylate the physiological substrate α-glutathionyl-ß-hydroxypropiovanillone, argues for broadening the definition of the omega class.

Characterization of a Phanerochaete chrysosporium glutathione transferase reveals a novel structural and functional class with ligandin properties.

Glutathione S-transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. A new fungal specific class of GST has been highlighted by genomic approaches. The biochemical and structural characterization of one isoform of this class in Phanerochaete chrysosporium revealed original properties. The three-dimensional structure showed a new dimerization mode and specific features by comparison with the canonical GST structure. An additional ß-hairpin motif in the N-terminal domain prevents the formation of the regular GST dimer and acts as a lid, which closes upon glutathione binding. Moreover, this isoform is the first described GST that contains all secondary structural elements, including helix α4' in the C-terminal domain, of the presumed common ancestor of cytosolic GSTs (i.e. glutaredoxin 2). A sulfate binding site has been identified close to the glutathione binding site and allows the binding of 8-anilino-1-naphtalene sulfonic acid. Competition experiments between 8-anilino-1-naphtalene sulfonic acid, which has fluorescent properties, and various molecules showed that this GST binds glutathionylated and sulfated compounds but also wood extractive molecules, such as vanillin, chloronitrobenzoic acid, hydroxyacetophenone, catechins, and aldehydes, in the glutathione pocket. This enzyme could thus function as a classical GST through the addition of glutathione mainly to phenethyl isothiocyanate, but alternatively and in a competitive way, it could also act as a ligandin of wood extractive compounds. These new structural and functional properties lead us to propose that this GST belongs to a new class that we name GSTFuA, for fungal specific GST class A.

Functional diversification of fungal glutathione transferases from the ure2p class.

The glutathione-S-transferase (GST) proteins represent an extended family involved in detoxification processes. They are divided into various classes with high diversity in various organisms. The Ure2p class is especially expanded in saprophytic fungi compared to other fungi. This class is subdivided into two subclasses named Ure2pA and Ure2pB, which have rapidly diversified among fungal phyla. We have focused our analysis on Basidiomycetes and used Phanerochaete chrysosporium as a model to correlate the sequence diversity with the functional diversity of these glutathione transferases. The results show that among the nine isoforms found in P. chrysosporium, two belonging to Ure2pA subclass are exclusively expressed at the transcriptional level in presence of polycyclic aromatic compounds. Moreover, we have highlighted differential catalytic activities and substrate specificities between Ure2pA and Ure2pB isoforms. This diversity of sequence and function suggests that fungal Ure2p sequences have evolved rapidly in response to environmental constraints.

Imaging of the most frequent superficial soft-tissue sarcomas.

Superficial soft-tissue sarcomas are malignant mesenchymal tumors located within the cutaneous and/or subcutaneous layers. Most superficial soft-tissue sarcomas are low-grade tumors; yet, the risk of local recurrence is high, and initial wide surgery is the main prognostic factor. Some of these superficial sarcomas may grow, following an infiltrative pattern, and their real extent may be underestimated clinically. Imaging techniques are useful to determine precisely the real margins of the tumor, especially in cases of clinically doubtful or recurrent or large superficial lesions. Imaging tools enable one to determine the relationship with the superficial fascia separating the subcutaneous layer from the underlying muscle. In our institution ultrasonographic examination is followed by magnetic resonance (MR) imaging when the size of the lesion exceeds 3-5 cm. Imaging assessment is performed prior to biopsy, enabling optimal surgical management. Imaging features of the main superficial sarcomas are detailed in the following article, according to their major locations: those arising in the epidermis and/or dermis, which are most often diagnosed by dermatologists, and the subcutaneous sarcomas.

Glutathione transferases of Phanerochaete chrysosporium: S-glutathionyl-p-hydroquinone reductase belongs to a new structural class.

The white rot fungus Phanerochaete chrysosporium, a saprophytic basidiomycete, possesses a large number of cytosolic glutathione transferases, eight of them showing similarity to the Omega class. PcGSTO1 (subclass I, the bacterial homologs of which were recently proposed, based on their enzymatic function, to constitute a new class of glutathione transferase named S-glutathionyl-(chloro)hydroquinone reductases) and PcGSTO3 (subclass II related to mammalian homologs) have been investigated in this study. Biochemical investigations demonstrate that both enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteinyl residue. This reaction leads to the formation of a disulfide bridge between the conserved cysteine and the removed glutathione from their substrate. The substrate specificity of each isoform differs. In particular PcGSTO1, in contrast to PcGSTO3, was found to catalyze deglutathionylation of S-glutathionyl-p-hydroquinone substrates. The three-dimensional structure of PcGSTO1 presented here confirms the hypothesis that it belongs not only to a new biological class but also to a new structural class that we propose to name GST xi. Indeed, it shows specific features, the most striking ones being a new dimerization mode and a catalytic site that is buried due to the presence of long loops and that contains the catalytic cysteine.

The fungal glutathione S-transferase system. Evidence of new classes in the wood-degrading basidiomycete Phanerochaete chrysosporium.

The recent release of several basidiomycete genome sequences allows an improvement of the classification of fungal glutathione S-transferases (GSTs). GSTs are well-known detoxification enzymes which can catalyze the conjugation of glutathione to non-polar compounds that contain an electrophilic carbon, nitrogen, or sulfur atom. Following this mechanism, they are able to metabolize drugs, pesticides, and many other xenobiotics and peroxides. A genomic and phylogenetic analysis of GST classes in various sequenced fungi--zygomycetes, ascomycetes, and basidiomycetes--revealed some particularities in GST distribution, in comparison with previous analyses with ascomycetes only. By focusing essentially on the wood-degrading basidiomycete Phanerochaete chrysosporium, this analysis highlighted a new fungal GST class named GTE, which is related to bacterial etherases, and two new subclasses of the omega class GSTs. Moreover, our phylogenetic analysis suggests a relationship between the saprophytic behavior of some fungi and the number and distribution of some GST isoforms within specific classes.

AtHMA3, a P1B-ATPase allowing Cd/Zn/Co/Pb vacuolar storage in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) Heavy Metal Associated3 (AtHMA3) protein belongs to the P1B-2 subgroup of the P-type ATPase family, which is involved in heavy metal transport. In a previous study, we have shown, using heterologous expression in the yeast Saccharomyces cerevisiae, that in the presence of toxic metals, AtHMA3 was able to phenotypically complement the cadmium/lead (Cd/Pb)-hypersensitive strain ycf1 but not the zinc (Zn)-hypersensitive strain zrc1. In this study, we demonstrate that AtHMA3 in planta is located in the vacuolar membrane, with a high expression level in guard cells, hydathodes, vascular tissues, and the root apex. Confocal imaging in the presence of the Zn/Cd fluorescent probe BTC-5N revealed that AtHMA3 participates in the vacuolar storage of Cd. A T-DNA insertional mutant was found more sensitive to Zn and Cd. Conversely, ectopic overexpression of AtHMA3 improved plant tolerance to Cd, cobalt, Pb, and Zn; Cd accumulation increased by about 2- to 3-fold in plants overexpressing AtHMA3 compared with wild-type plants. Thus, AtHMA3 likely plays a role in the detoxification of biological (Zn) and nonbiological (Cd, cobalt, and Pb) heavy metals by participating in their vacuolar sequestration, an original function for a P1B-2 ATPase in a multicellular eukaryote.

Comparison of the thiol-dependent antioxidant systems in the ectomycorrhizal Laccaria bicolor and the saprotrophic Phanerochaete chrysosporium.

Sequencing of the Laccaria bicolor and Phanerochaete chrysosporium genomes, together with the availability of many fungal genomes, allow careful comparison to be made of these two basidiomycetes, which possess a different way of life (either symbiotic or saprophytic), with other fungi. Central to the antioxidant systems are superoxide dismutases, catalases and thiol-dependent peroxidases (Tpx). The two reducing systems (thioredoxin (Trx) and glutathione/glutaredoxin (Grx)) are of particular importance against oxidative insults, both for detoxification, through the regeneration of thiol-peroxidases, and for developmental, physiological and signalling processes. Among those thiol-dependent antioxidant systems, special emphasis is given to the redoxin and methionine sulfoxide reductase (Msr) multigenic families. The genes coding for these enzymes were identified in the L. bicolor and P. chrysosporium genomes, were correctly annotated, and the gene content, organization and distribution were compared with other fungi. Expression of the Laccaria genes was also compiled from microarray data. A complete classification, based essentially on gene structure, on phylogenetic and sequence analysis, and on existing experimental data, was proposed. Comparison of the gene content of fungi from all phyla did not show huge differences for multigenic families in the reactive oxygen species (ROS) detoxification network, although some protein subgroups were absent in some fungi.

Characterization and regulation of PiDur3, a permease involved in the acquisition of urea by the ectomycorrhizal fungus Paxillus involutus.

Urea, which is known to be a source of nitrogen for the growth of many organisms, represents an important fertilizer in forest soils. Since most trees form symbiotic associations with ectomycorrhizal fungi, the capacities of these symbionts to take up and assimilate urea would determine the efficiency of urea nitrogen salvaging by plants. We showed that Paxillusinvolutus, an ectomycorrhizal basidiomycete, is capable of using urea as sole nitrogen source. We report the molecular characterization of an active urea transporter (PiDur3) isolated from this fungus. We demonstrated that the import of urea is a minor event on ammonium condition, since the expression of PiDUR3 is repressed by the high intracellular glutamine pool. Interestingly, on urea nutritive condition, the uptake of urea is rather mediated by the intracellular urea pool and particularly by urease efficiency.

CARM1 promotes adipocyte differentiation by coactivating PPARgamma.

The coactivator-associated arginine methyltransferase 1 (CARM1) is recruited to gene promoters by many transcription factors. To identify new pathways that use CARM1, we carried out a comprehensive transcriptome analysis of CARM1-knockout embryos. By using complementary DNA microarrays and serial analysis of gene expression, we identified various genes involved in lipid metabolism that were underrepresented in CARM1-knockout embryos, indicating an important role for this coactivator in adipose tissue biology. We also observed that the amount of brown fat in CARM1-knockout embryos is reduced. Furthermore, cells lacking CARM1 have a severely curtailed potential to differentiate into mature adipocytes. Reporter experiments and chromatin immunoprecipitation analysis show that CARM1 regulates these processes by acting as a coactivator for peroxisome proliferator-activated receptor gamma (PPARgamma). Together, these results show that CARM1 promotes adipocyte differentiation by coactivating PPARgamma-mediated transcription and thus might be important in energy balance.

Early rheumatoid arthritis: a review of MRI and sonographic findings.

OBJECTIVE: The introduction of anti-tumor necrosis factor alpha agents has opened new prospects in therapeutic management of patients with early rheumatoid arthritis, thereby creating new demands on radiologists to identify patients with aggressive disease at an early stage. As a result, imaging techniques such as MRI and sonography have developed during the past few years. CONCLUSION: This article illustrates the imaging findings that may be encountered with these techniques in patients with early rheumatoid arthritis.

Imaging features of musculoskeletal involvement in systemic sclerosis.

This article describes the radiographic, sonographic and magnetic resonance (MR) features of musculoskeletal involvement in patients with systemic sclerosis (SSc). Conventional radiography is the traditional method of detecting digital calcifications, but ultrasonography (US) is also able to detect such calcifications before they appear on radiographs. MR imaging can be used to diagnose overlapping conditions (i.e., SSc and myositis or SSc and rheumatoid arthritis), and less frequently, to reveal neurologic complications of SSc. In patients with vascular ulcers, MR angiography is able to depict decreased flow within collateral digital arteries.

Peptide uptake in the ectomycorrhizal fungus Hebeloma cylindrosporum: characterization of two di- and tripeptide transporters (HcPTR2A and B).

Constraints on plant growth imposed by low availability of nitrogen are a characteristic feature of ecosystems dominated by ectomycorrhizal plants. Ectomycorrhizal fungi play a key role in the N nutrition of plants, allowing their host plants to access decomposition products of dead plant and animal materials. Ectomycorrhizal plants are thus able to compensate for the low availability of inorganic N in forest ecosystems. The capacity to take up peptides, as well as the transport mechanisms involved, were analysed in the ectomycorrhizal fungus Hebeloma cylindrosporum. The present study demonstrated that H. cylindrosporum mycelium was able to take up di- and tripeptides and use them as sole N source. Two peptide transporters (HcPTR2A and B) were isolated by yeast functional complementation using an H. cylindrosporum cDNA library, and were shown to mediate dipeptide uptake. Uptake capacities and expression regulation of both genes were analysed, indicating that HcPTR2A was involved in the high-efficiency peptide uptake under conditions of limited N availability, whereas HcPTR2B was expressed constitutively.

NADP-dependent glutamate dehydrogenase: a dispensable function in ectomycorrhizal fungi.

There is much controversy on the contribution of NADP-dependent glutamate dehydrogenase (NADP-GDH) in NH4+ assimilation in ectomycorrhizal (ECM) fungi and ectomycorrhizas. Experiments reported here provide information on the dispensability of NADP-GDH in various ectomycorrhizal isolates. Glutamate dehydrogenase and glutamine synthetase (GS) enzyme activities were measured on mycelia grown under various nitrogen (N) conditions. The contribution of GDH in ammonium assimilation was further estimated by following 15N incorporation from (15NH4)2SO4 into glutamate, when GS was inhibited by phosphinothricin. Finally, gene amplification on cDNA and genomic DNA was performed using degenerated primers. Two groups of fungi could be distinguished. The GDH+ fungi include Hebeloma cylindrosporum-like fungi, which possess a functional NADP-GDH. The GDH- fungi include Paxillus involutus-like fungi for which the NADP-GDH activity, as well as the GDHA transcripts, were not detected, whatever the growth condition. All the results are consistent with the dispensability of the NADP-GDH function in ECM fungi, suggesting a minor role in ammonium assimilation in ectomycorrhizal fungi. We hypothesize that the lack of a functional NADP-GDH could be an evolutive adaptation in relation to the ecological niche of ECM fungi, rather than a transitional regulation in response to changes in N contents of the extracellular medium.

Ablation of the Sam68 RNA binding protein protects mice from age-related bone loss.

The Src substrate associated in mitosis of 68 kDa (Sam68) is a KH-type RNA binding protein that has been shown to regulate several aspects of RNA metabolism; however, its physiologic role has remained elusive. Herein we report the generation of Sam68-null mice by homologous recombination. Aged Sam68-/- mice preserved their bone mass, in sharp contrast with 12-month-old wild-type littermates in which bone mass was decreased up to approximately 75%. In fact, the bone volume of the 12-month-old Sam68-/- mice was virtually indistinguishable from that of 4-month-old wild-type or Sam68-/- mice. Sam68-/- bone marrow stromal cells had a differentiation advantage for the osteogenic pathway. Moreover, the knockdown of Sam68 using short hairpin RNA in the embryonic mesenchymal multipotential progenitor C3H10T1/2 cells resulted in more pronounced expression of the mature osteoblast marker osteocalcin when differentiation was induced with bone morphogenetic protein-2. Cultures of mouse embryo fibroblasts generated from Sam68+/+ and Sam68-/- littermates were induced to differentiate into adipocytes with culture medium containing pioglitazone and the Sam68-/- mouse embryo fibroblasts shown to have impaired adipocyte differentiation. Furthermore, in vivo it was shown that sections of bone from 12-month-old Sam68-/- mice had few marrow adipocytes compared with their age-matched wild-type littermate controls, which exhibited fatty bone marrow. Our findings identify endogenous Sam68 as a positive regulator of adipocyte differentiation and a negative regulator of osteoblast differentiation, which is consistent with Sam68 being a modulator of bone marrow mesenchymal cell differentiation, and hence bone metabolism, in aged mice.

Arginine methylation regulates IL-2 gene expression: a role for protein arginine methyltransferase 5 (PRMT5).

Arginine methylation is a post-translational modification resulting in the generation of aDMAs (asymmetrical omega-NG, NG-dimethylated arginines) and sDMAs (symmetrical omega-NG, N'G-dimethylated arginines). The role of arginine methylation in cell signalling and gene expression in T lymphocytes is not understood. In the present study, we report a role for protein arginine methylation in regulating IL-2 (interleukin 2) gene expression in T lymphocytes. Leukaemic Jurkat T-cells treated with a known methylase inhibitor, 5'-methylthioadenosine, had decreased cytokine gene expression, as measured using an NF-AT (nuclear factor of activated T-cells)-responsive promoter linked to the luciferase reporter gene. Since methylase inhibitors block all methylation events, we performed RNA interference with small interfering RNAs against the major PRMT (protein arginine methyltransferases) that generates sDMA (PRMT5). The dose-dependent decrease in PRMT5 expression resulted in the inhibition of both IL-2- and NF-AT-driven promoter activities and IL-2 secretion. By using an sDMA-specific antibody, we observed that sDMA-containing proteins are directly associated with the IL-2 promoter after T-cell activation. Since changes in protein arginine methylation were not observed after T-cell activation in Jurkat and human peripheral blood lymphocytes, our results demonstrate that it is the recruitment of methylarginine-specific protein(s) to cytokine promoter regions that regulates their gene expression.