Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita. The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro. Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development. IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.

Bioactive peptide natural products are an important source of therapeutics. Prominent examples are the 19 antibiotic penicillin and the immunosuppressant cyclosporine which are both produced by fungi and have 20 revolutionized modern medicine. Peptide biosynthesis can occur either non-ribosomally via large enzymes 21 referred to as non-ribosomal peptide synthetases (NRPS) or ribosomally. Ribosomal peptides are synthesized as 22 part of a larger precursor peptide where they are posttranslationally modified and subsequently proteolytically 23 released. Such peptide natural products are referred to as ribosomally synthesized and posttranslationallly 24 modified peptides (RiPPs). Their biosynthetic pathways have recently received a lot of attention, both from a 25 basic and applied research point of view, due to the discoveries of several novel posttranslational modifications 26 of the peptide backbone. Some of these modifications were so far only known from NRPSs and significantly 27 increase the chemical space covered by this class of peptide natural products. Latter feature, in combination with 28 the promiscuity of the modifying enzymes and the genetic encoding of the peptide sequence, makes RiPP 29 biosynthetic pathways attractive for synthetic biology approaches to identify novel peptide therapeutics via 30 screening of de novo generated peptide libraries and, thus, exploit bioactive peptide natural products beyond 31 their direct use as therapeutics. This review focuses on the recent discovery and characterization of novel RiPP 32 biosynthetic pathways in fungi and their possible application for the development of novel peptide therapeutics. 33 34 38 Meanwhile, the search for novel natural products with pharmacological activities continues. High-throughput 39 screens using molecular and cellular bioassays allow the evaluation of large numbers of natural products and 40 synthetically generated compound libraries for bioactivities of interest. Statistical analysis of the 41 complementarity of natural products and synthetic compounds showed that 40% of chemical scaffolds of natural 42 products are absent in synthetic compound libraries (Henkel et al. 1999), demonstrating that the biomedical 43 relevance of natural products lies in their wide range of structural diversity and complexity. Thus, natural 44 products continue to play a significant role in drug development. From the 1940s to 2014, 40% of small molecules 45 approved for cancer treatment were natural products or derivatives thereof (Newman and Cragg 2016). Plants 46 and microorganisms are the main sources of bioactive natural products (Dias et al. 2012). The vast dimension of 47 'microbial dark matter' suggested by genomic microbiome studies promises an enormous variety of bioactive48natural products yet to be discovered (Solden et al. 2016). This is also true for fungi where it is assumed that only 49 about one-twentieth of all existing fungi have been described and an even smaller fraction has been cultured 50 successfully in the lab (Jiang and An 2000...

Lectins are non-immunoglobulin-type proteins that bind to specific carbohydrate epitopes and play important roles in intra- and inter-organismic interactions. Here, we describe a novel fucose-specific lectin, termed CML1, which we identified from fruiting body extracts of Coprinopsis cinerea. For further characterization, the coding sequence for CML1 was cloned and heterologously expressed in Escherichia coli. Feeding of CML1-producing bacteria inhibited larval development of the bacterivorous nematode Caenorhabditis tropicalis, but not of C. elegans. The crystal structure of the recombinant protein in its apo-form and in complex with H type I or Lewis A blood group antigens was determined by X-ray crystallography. The protein folds as a sandwich of two antiparallel β-sheets and forms hexamers resulting from a trimer of dimers. The hexameric arrangement was confirmed by small-angle scattering of X-rays (SAXS). One carbohydrate binding site per protomer was found at the dimer interface with both protomers contributing to ligand binding, resulting in a hexavalent lectin. In terms of lectin activity of recombinant CML1, substitution of the carbohydrate-interacting residues His54, Asn55, Trp94 and Arg114 by Ala abolished carbohydrate-binding and nematotoxicity. While no similarities to any characterized lectin were found, sequence alignments identified many non-characterized agaricomycete proteins. These results suggest that CML1 is the founding member of a novel family of fucoside-binding lectins involved in the defense of agaricomycete fruiting bodies against predation by fungivorous nematodes.

Motor function and motor excitability can be modulated by changes of somatosensory input. Here, we performed a randomized single-blind trial to investigate behavioral and neurophysiological changes during temporary deafferentation of left upper arm and forearm in 31 right-handed healthy adults. Lidocaine cream was used to anesthetize the skin from wrist to shoulder, sparing the hand. As control condition, on a different day, a neutral cream was applied to the same skin area. The sequence (first Lidocaine, then placebo or vice versa) was randomized. Behavioral measures included the Grating Orientation Task, the Von Frey hair testing and the Nine-hole-peg-test. Transcranial magnetic stimulation was used to investigate short-interval intracortical inhibition, stimulus response curves, motor evoked potential amplitudes during pre-innervation and the cortical silent period (CSP). Recordings were obtained from left first dorsal interosseous muscle and from left flexor carpi radialis muscle. During deafferentation, the threshold of touch measured at the forearm was significantly worse. Other behavioral treatment-related changes were not found. The CSP showed a significant interaction between treatment and time in first dorsal interosseous muscle. CSP duration was longer during Lidocaine application and shorter during placebo exposure. We conclude that, in healthy subjects, temporary cutaneous deafferentation of upper and lower arm may have minor effects on motor inhibition, but not on sensory or motor function for the adjacent non-anesthetized hand.