Nuclear RNA foci from C9ORF72 expansion mutation form paraspeckle-like bodies.

The GGGGCC (G4C2) repeat expansion mutation in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (G4C2) n repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA hLinc-p21. As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (G4C2) n RNA foci. Overexpression of (G4C2)72 RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (G4C2)72 RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA NEAT1 Moreover, the knockdown of SFPQ protein in C9ORF72 expansion mutation-positive fibroblasts significantly reduces the number of (G4C2) n RNA foci. In conclusion, (G4C2) n RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation.

Medicinal Properties of the Genus Clitocybe and of Lectins from the Clouded Funnel Cap Mushroom, C. nebularis (Agaricomycetes): A Review.

Current knowledge of the medicinal properties of Basidiomycetes mushroom species of the genus Clitocybe and of the biological activity of C. nebularis fruiting bodies is reviewed. The main focus is the therapeutic potential of lectins from C. nebularis. Species of the genus Clitocybe, including C. nebularis, have not been traditionally considered as medicinal mushrooms; however, recent studies have demonstrated their antitumor, immunomodulatory, and antioxidative properties, their antimicrobial (antiviral, antibacterial, and antifungal) activities against various bacteria and fungi, as well as their potential use in therapy for alcoholism and as psychotropic agents. These activities have been shown to be due to various compounds, either isolated or in extracts, mainly polysaccharides but also phenols, ribonucleosides, and proteins. These include laccase, protease inhibitors, and lectins. C. nebularis has been shown to be rich in a variety of lectins and isolectins with distinct carbohydrate-binding specificities, showing versatile biological activities. They exhibit immunostimulatory and adhesion-/phagocytosis-promoting properties, as well as toxicity in various invertebrates. Mushroom species of the genus Clitocybe, including C. nebularis, thus constitute a valuable source of compounds showing diverse biological activities with a broad potential for applications in biomedicine or biotechnology. On the basis of such evidence reviewed here, we propose that C. nebularis and other Clitocybe species can be considered to be medicinal mushrooms.

ß-Trefoil structure enables interactions between lectins and protease inhibitors that regulate their biological functions.

Fungal ricin B-like lectins and protease inhibitors, mycocypins and mycospins, are important mediators in fungal defence against antagonists and all possess the ß-trefoil fold. We demonstrate here that fungal ß-trefoil proteins interact with each other, in addition to their apparent targets, and that these interactions modulate their biological activity. Such regulation of carbohydrate binding or inhibitory activity is observed for the first time in ß-trefoil proteins and could constitute a mechanism for regulating their physiological functions. It could also have implications in molecular recognition of other combinations of ß-trefoil proteins in other species.

Characterization of DNA G-quadruplex species forming from C9ORF72 G4C2-expanded repeats associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration.

The G4C2 hexanucleotide repeat expansion, located in the first intron of the C9ORF72 gene, represents a major genetic hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several hypotheses have been proposed on how the transcribed repeat RNA leads to the development of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. However, despite their importance, factors affecting the transcription of expanded-repeat RNA are not well known. As transcription is dependent on the DNA containing the expanded repeats, it is crucial to understand its structure. G-quadruplexes are known to affect expression on the level of DNA, therefore whether they form on the expanded-repeat DNA constitutes an important biological question. Using nuclear magnetic resonance and circular dichroism spectroscopy we show that DNA G4C2 with varying number of repeats d(G4C2)n form planar guanine quartets characteristic of G-quadruplexes. Additionally, we show DNA G-quadruplexes can form inter- and intra-molecularly in either parallel or anti-parallel orientation, based on d(G4C2) sequence length. This potential structural heterogeneity of longer disease-relevant repeats should therefore be taken into account when studying their role in disease pathogenesis.

A novel ß-trefoil lectin from the parasol mushroom (Macrolepiota procera) is nematotoxic.

UNLABELLED: Lectins are carbohydrate-binding proteins present in all organisms. Some cytoplasmic lectins from fruiting bodies of dikaryotic fungi are toxic against a variety of parasites and predators. We have isolated, cloned and expressed a novel, single domain lectin from Macrolepiota procera, designated MpL. Determination of the crystal structure revealed that MpL is a ricin B-like lectin with a ß-trefoil fold. Biochemical characterization, site-directed mutagenesis, co-crystallization with carbohydrates, isothermal titration calorimetry and glycan microarray analyses show that MpL forms dimers with the carbohydrate-binding site at the α-repeat, with the highest specificity for terminal N-acetyllactosamine and other ß-galactosides. A second putative carbohydrate-binding site with a low affinity for galactose is present at the γ-repeat. In addition, a novel hydrophobic binding site was detected in MpL with specificity for molecules other than carbohydrates. The tissue specific distribution of MpL in the stipe and cap tissue of fruiting bodies and its toxicity towards the nematode Caenorhabditis elegans indicate a function of MpL in protecting fruiting bodies against predators and parasites. DATABASE: Nucleotide sequence data have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers HQ449738 and HQ449739. Structural data have been deposited in the Protein Data Bank under accession codes 4ION, 4IYB, 4IZX and 4J2S.

Inhibition of the growth of colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom.

Proteins from higher fungi have attracted interest because of their exceptional characteristics. Macrocypins, cysteine protease inhibitors from the parasol mushroom Macrolepiota procera , were evaluated for their adverse effects and their mode of action on the major potato pest Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). They were shown to reduce larval growth when expressed in potato or when their recombinant analogues were added to the diet. Macrocypins target a specific set of digestive cysteine proteases, intestains. Additionally, protein-protein interaction analysis revealed potential targets among other digestive enzymes and proteins related to development and primary metabolism. No effect of dietary macrocypins on gene expression of known adaptation-related digestive enzymes was observed in CPB guts. Macrocypins are the first fungal protease inhibitors to be reported as having a negative effect on growth and development of CPB larvae and could also be evaluated as control agents for other pests.

Bivalent carbohydrate binding is required for biological activity of Clitocybe nebularis lectin (CNL), the N,N'-diacetyllactosediamine (GalNAcß1-4GlcNAc, LacdiNAc)-specific lectin from basidiomycete C. nebularis.

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N'-diacetyllactosediamine (GalNAcß1-4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its ß-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency.

CNL, a ricin B-like lectin from mushroom Clitocybe nebularis, induces maturation and activation of dendritic cells via the toll-like receptor 4 pathway.

A novel lectin, isolated from the basidiomycete mushroom Clitocybe nebularis and termed C. nebularis lectin (CNL), exhibits an immunostimulatory effect on the most potent antigen-presenting cells, the dendritic cells (DCs). Treatment of human monocyte-derived DCs with CNL in doses from 1 to 10 µg/ml resulted in a dose-dependent induction of overall DC maturation characteristics. Exposure of DCs to CNL for 48 hr resulted in extensive up-regulation of co-stimulatory molecules CD80 and CD86, as well as of the maturation marker CD83 and HLA-DR molecules. Such CNL-matured DCs (CNL-DCs) were capable of inducing a T helper type 1-polarized response in naive CD4+ CD45RA+ T cells in 5-day allogeneic co-cultures. The allostimulatory potential of CNL-DCs was significantly increased relative to untreated controls, as was their capacity to produce several pro-inflammatory cytokines such as interleukin-6, interleukin-8 and tumour necrosis factor-α. By using a specific Toll-like receptor 4 (TLR4) signalling inhibitor, CLI-095, as well as Myd88 inhibitory peptide, we have shown that DC activation by CNL is completely dependent on the TLR4 activation pathway. Furthermore, activation of TLR4 by CNL was confirmed via TLR4 reporter assay. Measurement of p65 nuclear factor-κB and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CNL stimulation of DCs revealed primarily an increase in nuclear factor-κB activity, with less effect on the induction of p38 MAPK signalling than of lipopolysaccharide-matured DCs. The CNL had the ability to activate human DCs in such a way as to subsequently direct T helper type 1 T-cell responses. Our results encourage the use of mushroom-derived lectins for use in therapeutic strategies with aims such as to strengthen anti-tumour immune responses.

Basidiomycete Clitocybe nebularis is rich in lectins with insecticidal activities.

Basidiomycete mushrooms are a rich source of unique substances, including lectins, that could potentially be useful in biotechnology or biomedical applications. Lectins are a group of carbohydrate-binding proteins with diverse biological activities and functions. Here, we demonstrate the presence of a number of lectins in the basidiomycete mushroom Clitocybe nebularis. Glucose-, galactose-, sucrose-, lactose-, and Sepharose-binding lectins were isolated from fruiting bodies using affinity chromatography on Sepharose-immobilized sugars or on Sepharose. The lectins were characterized biochemically and their binding specificities examined by agglutination and agglutination inhibition assays. In addition, insecticidal and anti-nutritional properties of the lectins were studied against a model organism, fruit fly (Drosophila melanogaster), and Colorado potato beetle (Leptinotarsa decemlineata). Of the several basidiomycete mushrooms screened, C. nebularis extract showed the most potent insecticidal activity. Sucrose-binding lectin showed the strongest activity against D. melanogaster, followed by lactose- and galactose-binding lectins. Feeding bioassays with Colorado potato beetle revealed that C. nebularis extract exhibited high anti-nutritional activity against the insect; and of those tested, only lactose-binding lectin, named CNL showed the effect. Mushroom C. nebularis is shown to be rich in a variety of lectins with versatile biological activities, including insecticidal and anti-nutritional effects. C. nebularis lectins could thus have potential for use as natural insecticides.

Purification, characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells.

BACKGROUND: Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions. METHODS: Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay. RESULTS: A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcalpha1-3(Fucalpha1-2)Galbeta-containing carbohydrates, and GalNAcbeta1-4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells. CONCLUSIONS: The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells. GENERAL SIGNIFICANCE: CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.

Ostreolysin enhances fruiting initiation in the oyster mushroom (Pleurotus ostreatus).

Fruiting initiation in mushrooms can be triggered by a variety of environmental and biochemical stimuli, including substances of natural or synthetic origin. In this work ostreolysin, a cytolytic protein specifically expressed during the formation of primordia and fruit bodies of Pleurotus ostreatus, was applied to nutrient media inoculated with mycelium of P. ostreatus, and its effects on mycelial growth and fructification of the mushroom studied. The addition of ostreolysin slightly inhibited the growth of mycelium, but strongly induced the formation of primordia, which appeared 10 d earlier than in control plates supplemented with bovine serum albumin or with the dissolving buffer alone. Moreover, ostreolysin stimulated the subsequent development of primordia into fruit bodies. However, direct involvement of this protein in the sporulation of the mushroom is unlikely, as it was also detected in large amounts in the non-sporulating strain of P. ostreatus.