1 eLS – New Article Template eLS 1 Fungal Ribotoxins 2 A27741 3 Lucía García-Ortega, Juan Palacios-Ortega and Álvaro Martínez-del-Pozo 4 Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, 5 Universidad Complutense, 28040 Madrid, Spain 6 Advanced article 7 Abstract: 8 Fungal ribotoxins constitute a family of extracellular ribonucleases with exquisite 9 specificity against rRNA. They induce apoptotic death of cells after inhibiting protein 10 translation. Ribosomes become functionally incompetent because ribotoxins cleave one 11 single phosphodiester bond, located at a unique and universally conserved loop, 12 needed for elongation factors function. As secreted proteins, ribotoxins need to cross 13 the membrane of their target cells in order to exert their catalytic activity and they do 14 it without receptor mediation. Using lipid model systems, it has been shown that they 15 are able to enter cells with membranes enriched in acidic phospholipids. Both 16 membrane-interacting and ribosomal-recognition activities are characterized by distinct 17 structural features. Even though the natural function of ribotoxins is not known yet, 18 their production by entomopathogenic fungi has suggested their insecticidal role. After 19 decades of detailed study, the biotechnological potential of ribotoxins in pest control 20 and as antitumor agents is becoming evident. 21 Key words: Antitumoral, elongation-factor, entomopathogen, fungal-toxin, 22 immunotoxin, insecticide, ribonuclease, ribosome, ribotoxin, sarcin. 23 Key Concepts: 24  Ribotoxins are extremely specific ribonucleases targeted against ribosomes 25  Ribotoxins are produced by fungi, some of them entomopathogens. 26  They show a high degree of structural conservation, including the local 27 arrangement of the active site residues 28  Cleavage of a single rRNA phosphodiester bond leads to cell death by inhibiting 29 translation 30  Ribotoxins are cyclizing RNases because they follow a general acid-base 31 mechanism with production of a 2’,3’-cyclic intermediate. 32 2 eLS – New Article Template  Ribotoxins must first enter their target cells to exert their lethal action. 1  Cell entrance is possible in cells with membranes enriched in acidic 2 phospholipids and altered permeability. 3  Ribotoxins are optimal candidates to be employed as pest control agents and in 4 antitumor immunotoxins. 5 Introduction 6 Ribotoxins are a group of extracellular and highly specific ribonucleases (RNases) 7 secreted by fungi (Lacadena et al., 2007, Olombrada et al., 2017a). Their name arises 8 from the fact that they have the ability to be extremely toxic by efficiently inactivating 9 ribosomes after cleaving a single phosphodiester bond located at a universally 10 conserved sequence (Schindler & Davies, 1977, Endo et al., 1983). This cleavage 11 produces the inactivation of the ribosomes leading to cell death by apoptosis (Olmo et 12 al., 2001). However, and given that they are extracellular proteins, they must first 13 enter the cells to exert their cytotoxic action. It is this entrance the rate limiting step of 14 ribotoxins’ action. There has not been found a protein receptor for ribotoxins which, 15 therefore, take advantage of permeability membrane changes produced by tumor 16 transformation, or virus infection, as well as their higher affinity for negatively charged 17 phospholipids (Gasset et al., 1989). This explains why α-sarcin, the most 18 representative member of the group, was originally discovered as an antitumor agent 19 (Olson & Goerner, 1965). Unfortunately α-sarcin was not as specific as desirable, 20 producing unwanted side-effects. Therefore, the research in this field was eventually 21 abandoned. It is now known, however, that ribotoxins constitute a more extended 22 family of proteins than initially described, with more variety of fungal origins and 23 sequences, but sharing key structural and enzymatic features which make them 24 optimum candidates to be employed in a different biotechnological approaches like 25 pest control and anticancer drugs development (Olombrada et al., 2014, Olombrada et 26 al., 2017a). 27 General structural features 28 All ribotoxins known are rather small proteins which share at least two different 29 elements of ordered secondary structure: A β-sheet, where the active site is located, 30 and a short α-helix (Figure 1). Interestingly, several other non-toxic fungal 31 extracellular RNases show identical three-dimensional arrangement including the 32 nature and geometrical disposition of the most important active-site residues (Figure 33 2). This explains why ribotoxins are considered the toxic representatives of a much 34 wider protein group, the RNase T1 family, which is one of the most deeply studied 35 proteins in history (Yoshida, 2001). Observation of their three-dimensional structures 36 explains their functional differences in terms of toxicity (Figure 1) since ribotoxins 37 display long non-ordered and positively charged loops, which are much shorter and 38 negatively charged in the non-toxic relatives. In fact, ribotoxins only share a maximum 39 3 eLS – New Article Template of 20% of their sequence with the other non-toxic RNases. These loops in ribotoxins 1 are responsible for recognizing the acid phospholipids which facilitate their cell entry 2 and also for specifically embracing the ribosomes to produce their highly specific and 3 lethal enzymatic cleavage (García-Ortega et al., 2002, García-Mayoral et al., 2005, 4 Álvarez-García et al., 2009). 5 Ribotoxins have been detected in many different fungi (Martínez-Ruiz et al., 6 1999), including entomopathogenic (Herrero-Galán et al., 2008, Olombrada et al., 7 2017b) and edible (Landi et al., 2017) species, but only the three-dimensional 8 structure of three of them has been solved so far: α-sarcin (Pérez-Cañadillas et al., 9 2000), restrictocin (Yang & Moffat, 1996) and hirsutellin A (HtA) (Viegas et al., 2009). 10 α-Sarcin and restrictocin show practically indistinguishable structures (Figure 1) as 11 expected from their higher than 85% sequence identity. On the other hand, HtA 12 displays unique features, starting with its size which is 20 amino acids shorter (130 13 against 150), though still larger than the non-toxic T1-like RNases (100-110 amino 14 acids). Moreover, HtA shows just 25% of sequence identity with the other larger 15 ribotoxins. Therefore, HtA structure contains non-ordered loops very different in 16 conformation and length while keeping the common central core characteristic of this 17 RNases family (Figure 1). Even so, it still conserves all functional features of ribotoxins. 18 These a priori exceptional features of the ribotoxin HtA seem to be now more common 19 with the recent discovery of anisoplin, a new ribotoxin, from Metarhizium anisopliae 20 with 70% sequence identity to HtA (Olombrada et al., 2017b). 21 Geometric arrangement of the active site residues 22 All ribotoxins show practically identical geometric disposition of their active-site 23 residues (Figure 2). This arrangement is also coincident with the one shown by RNase 24 T1, in good agreement with their common general acid-base catalytic mechanism 25 (Lacadena et al., 1998) (Figure 3). Accordingly, these RNases share at least four 26 amino acids located at strategic positions: Two histidines, one glutamic acid and one 27 arginine (His50, Glu96, His137, and Arg121, following α-sarcin numbering; Figure 2) 28 directly involved in the catalytic steps leading to the required proton transference to 29 cleave the bond (Figure 3) (Lacadena et al., 1999). They are located in the central β-30 sheet (Figure 2) with their side chains pointing towards the concave face of the protein 31 structure. This active site shows three highly representative features: (1) high density 32 of charged residues (Pérez-Cañadillas et al., 2000), (2) low surface accessibility of all 33 these titratable atoms and, consequently, (3) unusual pKa values of the catalytic Glu 34 and His residues (Pérez-Cañadillas et al., 1998), as well as unusual Nδ tautomeric 35 forms of the latter ones (Pérez-Cañadillas et al., 2003). 36 Another important residue in the active site is Tyr48 (α-sarcin numbering), 37 conserved in most of the members of the T1 family (Figure 2) and essential for α-38 sarcin full enzymatic activity (Álvarez-García et al., 2006). However, inspection of the 39 arrangement of the two smaller ribotoxins known so far (HtA and anisoplin) 40 4 eLS – New Article Template (Olombrada et al., 2017b), both produced by entomopathogenic fungi, shows that an 1 Asp residue appears at the equivalent position (Figure 2). Interestingly, mutagenic 2 analysis involving this strategic position has shown how these smaller versions display 3 a very different electrostatic arrangement (Herrero-Galán et al., 2012a, Maestro-López 4 et al., 2017), representing an optimum compromise among conformational freedom, 5 stability, specificity, and active-site plasticity. All these features together allow them to 6 accommodate the characteristic abilities of ribotoxins into a shorter and more stable 7 structure of intermediate size between that of the other nontoxic fungal RNases and 8 the previously known larger ribotoxins. 9 Enzymatic mechanism 10 Ribotoxins cleave RNA following a mechanism shared by all extracellular fungal RNases 11 characterized so far. Using dinucleosides, such as GpA, for example, it has been shown 12 how the hydrolysis of the 3’-5’ phosphodiester bond of these substrates takes place via 13 a 2’-3’ cyclic mononucleotide which is then converted to the corresponding 3’-14 monophosphate derivative as the final product of the reaction (Figure 3). Thus, 15 ribotoxins perform a general acid–base type endonucleolytic cleavage of RNA which fits 16 into a two-step mechanism, considered as the signature of cyclizing RNases (Lacadena 17 et al., 1998, Yoshida, 2001): A transphosphorylation reaction which is followed by the 18 hydrolysis of the mentioned cyclic intermediate (Figure 3). See also: Acid–Base 19 Catalysis by Enzymes (DOI: 10.1002/9780470015902.a0000602.pub2). 20 At least in α-sarcin, during the first step of the reaction Glu96 acts as the 21 general base and His137 as the general acid (Figure 3). The hydrolysis of the cyclic 22 derivative is then catalysed by the same groups, but playing opposite roles. It is now 23 well known that these α-sarcin His137 and Glu96 are the only residues that are 24 essential for performing the catalytic acid–base type reaction, though some other 25 mutants have been found to be inactive against the ribosome or an isolated mimetic 26 version of the targeted rRNA fragment, the sarcin-ricin loop or SRL (Lacadena et al., 27 1998, Lacadena et al., 1999). In fact, this Glu/His combination is the most common 28 pair of catalytic residues found in microbial RNases (Yoshida, 2001). The other His 29 residue, His50, is required in its protonated form to assist the electrostatic stabilization 30 of the transition state. Finally, the role of Arg121 has been studied with its 31 replacement by Gln or Lys. These mutations did not modify the conformation of the 32 protein, but abolished its ribosome inactivating activity (Lacadena et al., 2007). 33 Unexpectedly, these mutants were still active against a small and nonspecific substrate 34 such as ApA. Interestingly, the loss of the positive charge at that position produced 35 dramatic changes in α-sarcin’s ability to interact with phospholipid membranes 36 suggesting that proteins which have evolved to interact with nucleic acids, such as 37 RNases, would have developed structural determinants to recognize polyphosphate 38 lattices, such as cell membranes, which certainly can be considered as two-39 dimensional phosphate networks. 40 5 eLS – New Article Template The substrate 1 Ribotoxins specifically cleave a single phosphodiester bond within a universally 2 conserved rRNA sequence located in a key ribosomal structure known as the sarcin-3 ricin loop (SRL) (Figure 4). This name arises from the early observation that this SRL is 4 not only the target of fungal ribotoxins but also of the well-known family of ribosome-5 inactivating proteins (RIP), best represented by ricin (Stirpe, 2015). These RIP are also 6 highly specialized toxic proteins, produced by plants and fungi that inactivate 7 ribosomes by acting as N-glycosidases on the same unique rRNA structure as 8 ribotoxins do (Endo & Tsurugi, 1987, Correll et al., 1999). They depurinate a single 9 nucleotide contiguous to the phosphodiester bond cleaved by ribotoxins (Figure 4), 10 producing a very similar inactivating effect. Obviously, ribotoxins are also ribosome-11 inactivating proteins. However, there is a rather general consensus to employ this 12 name only for the N-glycosidases while the term ribotoxins refers only to the toxic 13 RNases of this review. See also: Ribonucleases (DOI: 10.1038/npg.els.0003895). 14 Cleavage of the large rRNA at the SRL leads to complete inactivation of the 15 ribosome because this loop interacts with translation factors that bind and exert their 16 essential function on the ribosome assisted by GTP hydrolysis (Nierhaus et al., 1992). 17 It has been precisely determined that it is elongation factor G (EF-G) binding the most 18 perturbed event by ribotoxins cleavage (García-Ortega et al., 2010). Binding is 19 strongly impaired and consequently GTP hydrolysis and mRNA–tRNA translocation 20 during elongation do no take place at a significant rate leading to dysfunctional 21 ribosomes. See also: Elongation Factors: Bacterial (DOI: 22 10.1038/npg.els.0003932), rRNA Structure (DOI: 10.1038/npg.els.0000537), and 23 Ribosome Structure and Shape (DOI: 10.1038/npg.els.0000534). 24 The positively charged surface of ribotoxins allows them to establish favourable 25 electrostatic interactions between their active site residues and the rRNA, explaining 26 their highly specific recognition of the SRL (García-Mayoral et al., 2005, Korennykh et 27 al., 2006, Álvarez-García et al., 2009). So far, the regions which are known to 28 participate in this interaction are the Lys‐rich region of loop 3, which would interact 29 with a phosphodiester bond around the bulged G of the SRL, and the stretch 30 comprising residues 51–55 of loop 2 which, altogether with some residues of loop 5, 31 would contact the GAGA tetra-loop that is cleaved by the toxin (Figure 4) (Yang et al., 32 2001, García-Mayoral et al., 2005). Docking models suggest other α‐sarcin regions 33 recognizing more ribosomal elements (García-Mayoral et al., 2005), a prediction that 34 would justify the different affinity shown by ribotoxins against ribosomes from different 35 species, in spite of the universal conservation of the SRL. For example, the α-sarcin 36 11-16 residues stretch would interact with ribosomal protein uL14, explaining why 37 deletion of the N-terminal β-hairpin renders an active but non-specific RNase unable to 38 unequivocally target the SRL (García-Ortega et al., 2002, García-Mayoral et al., 2004). 39 In addition, some other not yet detected ribosomal regions could also participate in 40 this specific recognition. Good candidates would be those ones involved in the 41 6 eLS – New Article Template recruitment of elongation factors during translation. That could have been the case of 1 the highly dynamic protruding structure of the ribosome that serves as an anchoring 2 platform for elongation factors, known as the ribosomal stalk, which has been shown 3 to fulfil this specific function for ricin, for example (Tumer & Li, 2012). Quite 4 surprisingly, given that ricin and α-sarcin share identical rRNA target, ribotoxins do not 5 seem to need to interact with the ribosomal stalk in order to reach the SRL (Olombrada 6 et al., 2014). It has to be then concluded that the search for key specific interactions 7 established between ribotoxins and ribosome from different origins is far from being 8 closed yet. 9 Crossing the membrane 10 As mentioned above, the toxicity of ribotoxins results from the combination of their 11 highly specific RNase activity and their ability to cross membranes. Given that no 12 protein receptor for ribotoxins has been found, the lipid composition of membranes 13 plays an important role in their cytotoxic specific activity. Using lipid model systems 14 has been shown that α‐sarcin interacts with lipid vesicles enriched in acidic 15 phospholipids, promoting vesicle aggregation. This event leads to vesicle fusion with 16 intermixing of phospholipids and leakage of their aqueous contents (Gasset et al., 17 1989, Gasset et al., 1990) (Figure 5). Within this idea, this protein has been also 18 proven to have that ability to translocate across a negatively charged bilayer in the 19 absence of any other protein component (Oñaderra et al., 1993). Interestingly, the 20 outer monolayer of tumor cell membranes appears to be enriched in negative 21 phospholipids. Quite surprisingly, however, this behaviour with model vesicles does not 22 seem to be strictly conserved among ribotoxins. Again, HtA is the known exception 23 because it does not promote vesicle aggregation even though it shows higher 24 membrane‐permeabilizing ability than α‐sarcin in leakage experiments and is still able 25 to penetrate its target cells with at least as much efficiency as α-sarcin (Herrero-Galán 26 et al., 2008). 27 The structural details of this ribotoxins-lipid interaction have also been 28 determined to great extent in α-sarcin. In this protein, the β-sheet region comprising 29 residues 116–139 seems to be a key element in the hydrophobic interaction with 30 membranes (Mancheño et al., 1995, Mancheño et al., 1998). Loop 3 Lys residues 111 31 and 114 (Figure 1) would also take part in the electrostatic interactions needed to 32 bring vesicles into contact (Castaño-Rodríguez et al., 2015) (Figure 5). On the other 33 hand, in the case of HtA a role in membrane‐permeabilizing activity has been assigned 34 to Trp 71 and 78 (Herrero-Galán et al., 2012b). Trp residues in α-sarcin, although 35 differently located in the structure, seem to play a very similar role too (De Antonio et 36 al., 2000) (Figure 5). This ability to interact with lipid membranes has also been 37 associated with the positively charged N‐terminal β‐hairpin of ribotoxins because its 38 deletion in α‐sarcin yields a non‐toxic but active ribonuclease with altered membrane 39 interaction properties (García-Ortega et al., 2002). Intriguingly, it is in this region 40 where HtA shows more variability when compared to α‐sarcin (Figure 1). The N‐41 7 eLS – New Article Template terminal β‐hairpin of HtA is much shorter, a difference which appears to be 1 compensated by the extension of loop 5, which also exhibits a higher amount of 2 positive charges (Herrero-Galán et al., 2012a). 3 Biological function and biotechnological applications 4 It is not clear why fungi secrete ribotoxins though they should have predating and/or 5 defensive functions. At least for Aspergillus, the main ribotoxin producer genus so far, 6 they seem to be produced during conidia maturation, most probably as a defence 7 mechanism against predators(Brandhorst et al., 1996).The discovery that the 8 entomopathogenic fungus H. thompsonii was synthesizing HtA (Herrero-Galán et al., 9 2008), followed by the recent characterization of anisoplin (Olombrada et al., 2017b), 10 a new small HtA-like ribotoxin produced by other entomopathogenic fungi such as 11 Metarhizium anisopliae, suggested the possibility of being insecticidal proteins. This 12 function was then proved for α-sarcin and some other ribotoxins such as HtA 13 (Olombrada et al., 2013, Olombrada et al., 2014, Olombrada et al., 2017a, Olombrada 14 et al., 2017b). 15 These results have opened a new biotechnological venture to use ribotoxins as 16 the base to design new and environmentally friendly bioinsecticides. In fact, resistance 17 to pesticides has increased over the years and, simultaneously, pest diseases are the 18 cause of up to 40% losses in agriculture production around the world. Some 19 entomopathogenic fungi, such as the ribotoxins producers H. thompsonii and M. 20 anisopliae, have been already commercialized as control agents to manage crop 21 diseases (Kanga et al., 2002). Accordingly, ribotoxins could be used independently or 22 as part of biopesticide formulas, being a more controlled and reproducible product than 23 the whole fungal extract (Olombrada et al., 2013, Olombrada et al., 2014, Olombrada 24 et al., 2017a). The potential toxicity of ribotoxins against vertebrates could be 25 overcome by the design of new variants with diminished non-specific toxicity (Herrero-26 Galán et al., 2012b). Finally, their combination with insect pathogenic viruses such as 27 some baculoviruses represents another promising approach for biocontrol. Natural 28 baculoviruses have been already used as effective biopesticides thanks to their 29 specificity, but their genetic modification to deliver ribotoxins seems to be an optimum 30 alternative for pest control (Olombrada et al., 2017a). 31 As mentioned at the beginning of this review, ribotoxins were first discovered as 32 antitumor agents. Unfortunately, further studies revealed an unspecific cytotoxicity 33 against non-tumor cells which discouraged their use in anticancer therapies. 34 Fortunately enough, the interest for ribotoxins has revived as part of antitumor 35 immunotoxins (Tomé-Amat et al., 2015a). They are chimeric molecules composed of a 36 specific antibody fragment, responsible for targeting a specific cell surface antigen, 37 linked to a ribotoxin moiety that promotes cell death. Immunotoxin designs based on 38 the employment of ribotoxins have been shown to be highly effective, with the 39 additional benefit of not showing any detectable undesirable side effect, most probably 40 8 eLS – New Article Template due to the high specific antigen recognition exerted by the employed antibody (Tomé-1 Amat et al., 2015a, Jones et al., 2016, Olombrada et al., 2017a). This approach has 2 been recently improved with the incorporation of different variants such as one that is 3 unable to cross membranes but still retains the ribonucleolytic activity (Tomé-Amat et 4 al., 2015b) or a deimmunized variant of α-sarcin showing a complete lack of T cell 5 activation in in vitro assays (Jones et al., 2016). 6 Acknowledgements 7 We truly thank all students and associates who have contributed to develop this field of 8 ribotoxins’ study along more than three decades, working not only as part of our Toxic 9 Proteins Group in Madrid (Spain), but also in many other laboratories around the 10 world. These contributions at the basic Science level are now beginning to pay off with 11 applications which eventually will be beneficial for Society. 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Toxicon 12 83: 69-74. 13 Olombrada M, Lázaro-Gorines R, López-Rodríguez JC, Martínez-Del-Pozo A, Oñaderra 14 M, Maestro-López M, Lacadena J, Gavilanes JG & García-Ortega L (2017a) Fungal 15 Ribotoxins: A Review of Potential Biotechnological Applications. Toxins 9: E71. 16 Olombrada M, Medina P, Budia F, Gavilanes JG, Martínez-Del-Pozo A & García-Ortega L 17 (2017b) Characterization of a new toxin from the entomopathogenic fungus 18 Metarhizium anisopliae: the ribotoxin anisoplin. Biological Chemistry 398: 135-142. 19 Olson BH & Goerner GL (1965) -Sarcin, a New Antitumor Agent. I. Isolation, 20 Purification, Chemical Composition, and the Identity of a New Amino Acid. Applied 21 Microbiology 13: 314-321. 22 Oñaderra M, Mancheño JM, Gasset M, Lacadena J, Schiavo G, Martínez-del-Pozo A & 23 Gavilanes JG (1993) Translocation of -sarcin across the lipid bilayer of asolectin 24 vesicles. Biochemical Journal 295: 221-225. 25 Pérez-Cañadillas JM, Campos-Olivas R, Lacadena J, Martínez-del-Pozo A, Gavilanes JG, 26 Santoro J, Rico M & Bruix M (1998) Characterization of pKa values and titration shifts 27 in the cytotoxic ribonuclease -sarcin by NMR. Relationship between electrostatic 28 interactions, structure, and catalytic function. Biochemistry 37: 15865-15876. 29 Pérez-Cañadillas JM, Santoro J, Campos-Olivas R, Lacadena J, Martínez-del-Pozo A, 30 Gavilanes JG, Rico M & Bruix M (2000) The highly refined solution structure of the 31 cytotoxic ribonuclease -sarcin reveals the structural requirements for substrate 32 recognition and ribonucleolytic activity. Journal of Molecular Biology 299: 1061-1073. 33 Pérez-Cañadillas JM, García-Mayoral MF, Laurents DV, Martínez-del-Pozo A, Gavilanes 34 JG, Rico M & Bruix M (2003) Tautomeric state of -sarcin histidines. N tautomers are 35 a common feature in the active site of extracellular microbial ribonucleases. FEBS 36 Letters 534: 197-201. 37 12 eLS – New Article Template Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC & Ferrin TE 1 (2004) UCSF Chimera--a visualization system for exploratory research and analysis. 2 Journal of Computational Chemistry 25: 1605-1612. 3 Schindler DG & Davies JE (1977) Specific cleavage of ribosomal RNA caused by -4 sarcin. Nucleic Acids Research 4: 1097-1110. 5 Stirpe F (2015) Ribosome-inactivating proteins: An overview. Plant Toxins 1-29. 6 Tomé-Amat J, Olombrada M, Ruiz-de-la-Herran J, Pérez-Gómez E, Andradas C, 7 Sánchez C, Martínez L, Martínez-del-Pozo A, Gavilanes JG & Lacadena J (2015a) 8 Efficient in vivo antitumor effect of an immunotoxin based on ribotoxin -sarcin in nude 9 mice bearing human colorectal cancer xenografts. Springerplus 4: 168. 10 Tomé-Amat J, Herrero-Galán E, Oñaderra M, Martínez-Del-Pozo A, Gavilanes JG & 11 Lacadena J (2015b) Preparation of an engineered safer immunotoxin against colon 12 carcinoma based on the ribotoxin hirsutellin A. FEBS Journal 282: 2131-2141. 13 Tumer NE & Li XP (2012) Interaction of ricin and Shiga toxins with ribosomes. Current 14 Topics in Microbiology and Immunology 357: 1-18. 15 Viegas A, Herrero-Galán E, Oñaderra M, Macedo AL & Bruix M (2009) Solution 16 structure of hirsutellin A. New insights into the active site and interacting interfaces of 17 ribotoxins. FEBS Journal 276: 2381-2390. 18 Yang X, Gerczei T, Glover L & Correll CC (2001) Crystal structures of restrictocin-19 inhibitor complexes with implications for RNA recognition and base flipping. Nature 20 Structural Biology 8: 968-973. 21 Yang XJ & Moffat K (1996) Insights into specificity of cleavage and mechanism of cell 22 entry from the crystal structure of the highly specific Aspergillus ribotoxin, restrictocin. 23 Structure 4: 837-852. 24 Yoshida H (2001) The ribonuclease T1 family. Methods in Enzymology 341: 28-41. 25 Further Reading: 26 Carreras-Sangrà N, Álvarez-García E, Herrero-Galán E, Tomé-Amat J, Lacadena J, 27 Alegre-Cebollada J, Oñaderra M, Gavilanes JG & Martínez-del-Pozo A (2008) The 28 therapeutic potential of fungal ribotoxins. Current Pharmaceutical Biotechnology 9: 153-29 160. 30 Carreras-Sangrà N, Tomé-Amat J, García-Ortega L, Batt CM, Oñaderra M, Martínez-del-31 Pozo A, Gavilanes J & Lacadena J (2012) Production and characterization of a colon 32 cancer specific immunotoxin based on the fungal ribotoxin α-sarcin. Protein Engineering 33 Design and Selection 25: 425-435. 34 García-Mayoral MF, Martínez-del-Pozo A, Campos-Olivas R, Gavilanes JG, Santoro J, Rico 35 M, Laurents DV & Bruix M (2006) pH-dependent conformational stability of the ribotoxin 36 α-sarcin and four active site charge substitution variants. Biochemistry 45: 13705–37 13 eLS – New Article Template 13718. 1 Gasset M, Mancheño JM, Lacadena J, Martínez-del-Pozo A, Oñaderra M & Gavilanes JG 2 (1995) Spectroscopic characterization of the alkylated α-sarcin cytotoxin: analysis of the 3 structural requirements for the protein–lipid bilayer hydrophobic interaction. Biochimica 4 et Biophysica Acta 1252: 43–52. 5 Herrero-Galán E, García-Ortega L, Olombrada M, Lacadena J, Martínez-del-Pozo A, 6 Gavilanes J & Oñaderra M (2013) Hirsutellin A: A paradigmatic example of the 7 insecticidal function of fungal ribotoxins. Insects 4: 339-356. 8 Martínez-Ruiz A, García-Ortega L, Kao R, Lacadena J, Oñaderra M, Mancheño JM, Davies 9 J, Martínez-del-Pozo A & Gavilanes JG (2001) RNase U2 and α-sarcin: A study of 10 relationships. Methods in Enzymology 341: 335–351. 11 Masip M, Lacadena J, Mancheño JM, Oñaderra M, Martínez-Ruiz A, Martínez-del-Pozo A 12 & Gavilanes JG (2001) Arginine 121 is a crucial residue for the specific cytotoxic activity 13 of the ribotoxin α-sarcin. European Journal of Biochemistry 268: 6190–6196. 14 Pérez-Cañadillas JM, Campos-Olivas R, Lacadena J, Martínez-del-Pozo A, Gavilanes JG, 15 Santoro J, Rico M & Bruix M (1998) Characterization of pKa values and titration shifts in 16 the cytotoxic ribonuclease a-sarcin by NMR. Relationship between electrostatic 17 interactions, structure, and catalytic function. Biochemistry 37: 15865–15876. 18 Glossary: 19 Baculovirus: Family of viruses which specifically infect invertebrate animals. Some are 20 so specific against its insect host that can be used biological agents in pest control. 21 They are also used to produce eukaryotic proteins in heterologous systems made of 22 insect cell lines. 23 Biopesticide: Pesticides derived from natural materials such as animals, plants or 24 microorganism and usually considered more environmentally friendly than the classical 25 pesticides of chemical synthesis origin. 26 Elongation-factor: Family of proteins which intervene in translational elongation 27 through interaction with specific regions of the ribosome. They are GTPases which use 28 the energy arising from GTP hydrolysis to facilitate the movement and turnover 29 required to elongate the polypeptide chain. 30 Entomopathogen: Any agent that is pathogenic to insects. 31 Glycosidase: Family of enzymes that catalyze the hydrolysis of glycosidic linkages. 32 Therefore they take part in degrading oligosaccharides and glycoconjugates. 33 Tautomer: Constitutional isomers of organic compounds that readily interconvert by 34 relocation of a proton. 35 36 14 eLS – New Article Template Figures: 1 2 3 4 5 6 7 8 9 10 Figure 1. Representation of the three-dimensional structure of representative 11 fungal RNases. Diagrams showing the three dimensional structure of ribotoxins α-12 sarcin (PDB ID: 1DE3), restrictocin (PDB ID: 1AQZ) and HtA (PDB ID: 2KAA), and two 13 non-toxic fungal extracellular RNases from the same family: RNases T1 (PDB ID: 14 9RNT) and U2 (PDB ID:1RTU) Diagrams were generated using the Chimera software 15 (Pettersen et al., 2004). 16 17 15 eLS – New Article Template 1 Figure 2. Representation of the active site arrangement of the most 2 representative fungal RNases. The catalytic triad made of two His and one Glu 3 residues is conserved in all proteins shown, as well as α-sarcin Arg121, while a fifth 4 residue, α-sarcin Leu145, maintains its highly hydrophobic character (Phe or Leu). The 5 position corresponding to α-sarcin Tyr48 is also conserved except for HtA and anisoplin 6 (not shown) where the equivalent position is occupied by an Asp residue (Asp40). 7 Diagrams were generated using the Chimera software (Pettersen et al., 2004). 8 9 16 eLS – New Article Template 1 17 eLS – New Article Template 1 Figure 3: Catalytic mechanism of cyclizing RNases. The catalytic mechanism of 2 cyclic RNases such as ribotoxins against a dinucleotide substrate (ApA or GpA) is 3 shown. A transphosphorylation process (in which the corresponding 2’,3’ cyclic 4 mononucleotide and adenosine are produced) is followed by hydrolysis of the cyclic 5 nucleotide to produce the corresponding 3’-mononucleotide. Side chains of residues 6 corresponding to α-sarcin His50, Glu96, and His137 are also shown, indicating at the 7 bottom left corner of the figure their spatial location in the context of the whole protein 8 three-dimensional structure. 9 10 18 eLS – New Article Template 1 2 3 4 Figure 4. The substrate of ribotoxins. (A) Three‐dimensional structure of the large 5 ribosomal subunit of Escherichia coli (PDB ID: 2AW4). The location of L1 and L7/L12 6 stalks (absent in this crystal) and E, P and A sites are indicated. Conserved proteins 7 around the SRL (orange) appear in different colors: uL6 (green), uL11 (red), and uL14 8 (blue). Other ribosomal proteins appear in light gray. 23S (dark gray) and 5S (cyan) 9 rRNAs are also shown; (B) SRL structure. The bulged G (red), the GAGA tetraloop 10 (blue), the bond cleaved by α‐sarcin and the adenine depurinated by ricin are 11 indicated. Diagrams were generated using the Chimera software (Pettersen et al., 12 2004). 13 14 19 eLS – New Article Template 1 Figure 5. Schematic representation of the translocation mechanism of α-2 sarcin acroos the bilayer of negatively charged phospholipid vesicles. (A) 3 Binding experiments reveal a strong ribotoxin–lipid vesicle interaction that causes 4 vesicle aggregation (B) mediated by the formation of a vesicle dimer maintained by 5 protein–protein associations. The N-terminal stretch as well as some of the positively 6 charged loops play a key role at this step. (C) Then, the β-sheet region comprising 7 residues 116–139, altogether with the Trp side-chains (in pink), establishes a 8 destabilizing hydrophobic interaction with the membrane which leads to (D) protein 9 internalization. 10