Plant -Thionins: Novel Insights on the Mechanism of Action of a Multi

The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253

Cells in focus Plant ␥-thionins: Novel insights on the mechanism of action of a multi-functional class of defense proteins Patr´ıcia B. Pelegrini, Octavio´ L. Franco ∗

Centro de Análises Proteômicas e Bioqu´ımicas, Pós-Gradua¸cão em Ciências Genômicas e Biotecnologia, Universidade Católica de Bras´ılia, SGAN Quadra 916, Módulo B, Av. W5 Norte 70.790-160 Asa Norte Bras´ılia/DF, Brazil Received 2 December 2004; received in revised form 13 May 2005

Abstract

This review focuses on the first plant defense protein class described in literature, with growth inhibition activity toward pathogens. These peptides were named ␥-thionins or defensins, which are small proteins that can be classified into four main subtypes according to their specific functions. ␥-Thionins are small cationic peptides with different and special abilities. They are able to inhibit digestive enzymes or act against bacteria and/or fungi. Current research in this area focuses particularly these two last targets, being the natural crop plant defenses improved through the use of transgenic technology. Here, we will compare primary and tertiary structures of ␥-thionins and also will analyze their similarities to scorpion toxins and insect defensins. This last comparison offers some hypothesis for ␥-thionins mechanisms of action against certain pathogens. This specific area has benefited from the recent determination of many ␥-thionin structures. Furthermore, we also summarize molecular interactions between plant ␥-thionins and fungi receptors, which include membrane proteins and lipids, shedding some light over pathogen resistance. Researches on ␥-thionins targets could help on plant genetic improvement for production of increased resistance toward pathogens. Thus, positive results recently obtained for transgenic plants and future prospects in the area are also approached. Finally, ␥-thionins activity has also been studied for future drug development, capable of inhibit tumor cell growth in human beings. © 2005 Published by Elsevier Ltd.

Keywords: ␥-Thionins; Defensins; Anti-fungal; Anti-bacterial; Enzyme inhibition; Cell death; Plant defense

Abbreviations: alfAFP, anti-fungal defensin from alfalfa; BPTI, bovine pancreatic trypsin inhibitor; BPT, bovine pancreatic trypsin; BCP-2, anti-microbial peptide from barley grain; CCA, Callosobruchus chinesis ␣-amylase; CMA, Callosobruchus maculatus ␣-amylase; Cp-thionin, cowpea thionin; CSH, cysteine stabilized helix motif; CS␣␤, cysteine-stabilized ␣␤ motif; DmAMP1, anti-microbial peptide from Dahlia merckii; Fa-AMP1, anti-microbial protein from Fagopyrum esculentum; HTH, helix turn helix motif; LPS, lipophospho surface; MsDef1, defensin from Medicago sativa; MtDef2, defensin from Medicago turcatula; NaD1, Nicotiana alata defensin; NMR, nuclear molecular resonance; Psd1, Defensin from Pisum sativum; PPA, porcine pancreas ␣-amylase; Pth-St1, pseudo-thionin from Solanum tuberosum 1; PT, Pyrularia thionin; ROI, reactive oxygen intermediates; SI␣1, ␣-amylase from sorghum inhibitor; SPE10, protein from Pachyrrihizus erosus; SNP, serine nonapeptide; TAD1, Triticum aestivum defensin 1; Tu-AMP, anti-microbial protein from Tulipa gesneriana; VaD1, protein from Vigna angularis ∗ Corresponding author. Tel.: +55 61 3448 7220; fax: +55 61 3347 4797. E-mail address: [email protected] (O.L. Franco).

1357-2725/$ – see front matter © 2005 Published by Elsevier Ltd. doi:10.1016/j.biocel.2005.06.011 2240 P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253

1. Introduction teins from types III and IV showed extreme toxicity to mammalian cell lines. However, this classification In the decade of 1960, a novel class of plant pep- is imprecise, since some ␥-thionin from types III and tides, which presented lethal in vitro activities against IV showed biological activity of groups I and II and plant pathogens were discovered and denominated ␥- vice versa (Selitrennikoff, 2001). Furthermore, this thionins. They are the first eukaryotic peptides puri- report will describe several plant ␥-thionins, in order fied, which presented a key role in plant defense to shed some light over this obscure question. Due to (Davis, Dulbecco, Eisen, Ginsberg, & Wood, 1968). these intrinsic characteristics described above, plant This peptide class, found in several plant tissues, ␥-thionins are attractive candidates for the control of as seeds, stems and roots, was located both inside pests and especially pathogens, been the use of plant and outside of cell, including extra cellular space ␥-thionins through plant genetic engineering focused (Selitrennikoff, 2001). They have also been named in this review. Structural ␥-thionins properties will be defensins by some authors due their high structural and surveyed and issues that affect their specificities of functional similarities (Lay, Schirra, Scanlon, Ander- interaction will be addressed. son, & Craik, 2003; Terras et al., 1995; Thevissen et al., 2004; Thomma, Cammue, & Thevissen, 2003a,b). ␥-Thionins consist of small basic peptides 2. Primary and tertiary structure analysis with approximately 45–47 residues long, in which 4–8 of these are cysteine residues that form disulfide bonds. As described before, ␥-thionins are small and stable Generally, ␥-thionins are studied in monomeric forms, proteins, composed by 45–47 amino acids residues, but they also are capable to form oligomers (Melo some of them positively charged, which give them a et al., 2002; Song et al., 2005). The three-dimensional cationic feature (Colilla, Rocher, & Mendez, 1990; Lay structure of several ␥-thionins have already been stud- & Anderson, 2005; Villa-Perello´ et al., 2003). Most ied in detail, both by X-ray crystallography as well residues are extremely variable, demonstrating by by NMR, where was observed a typical two-layer ␣␤ in silico studies, which primary structure homology sandwich that creates an amphipatic molecule (Bloch, of ␥-thionins is insufficient to determinate their bio- Patel, Baud, Zvelebil, & Carr, 1998; Romagnoli et al., logical function (de Lucca & Walsh, 1999; Selitren- 2003; Villa-Perello,´ Sanchez-Vallet,´ Garc´ıa-Olmedo, nikoff, 2001). An exception occurs with half-cystines Molina, & Andeu, 2003). They appear to play diverse involved in S S bonds establishment that are extremely roles in nature, showing anti-bacterial and/or anti- conserved (Fig. 1)(Li et al., 2002; Mendez et al., fungal activity (Terras et al., 1993; Thevissen et al., 1990; Nitti et al., 1995; Orru et al., 1997). However, 1996), the ability to inhibit mammalian cell growth by it has been found that ␥-thionins C-termini domain membrane permeabilization (Li et al., 2002) and the is an important determinant on anti-fungal activity, capability of inhibit insect ␣-amylases and proteinases as well basic amino acid, such as lysine and arginine (Bloch & Richardson, 1991; Melo et al., 2002). Despite (Fig. 1A) (Spelbrink et al., 2004). Hence, other studies that plant ␥-thionins act on plant defense against pests have demonstrated a conserved bioactive structure and pathogens, some exceptions were observed. One between plant anti-microbial peptides (Villa-Perello,´ of them, known as crambin, a neutrally charged pro- Sanchez-Vallet,´ Garc´ıa-Olmedo, Molina, & Andreu, tein from Crambe abyssinica seeds, neither showed 2005). When NaD1, a ␥-thionin-like protein from any anti-microbial activity nor enzyme inhibition, but Nicotiana alata flowers with biological activity is responsible for a sweet taste in plant seeds (Jelsch toward insect-pests, was compared to other sequences et al., 2000; Lobb et al., 1996; Schrader-Fischer & from data bank (NCBI), an enhanced similarity was Apel, 1994; Yamano & Teeter, 1994; Yamano, Heo, observed to alfAFP,an anti-fungal defensin from alfalfa & Teeter, 1997). ␥-Thionins are classified into a super (Medicago sativa)(Lay et al., 2003). Otherwise, NaD1 family divided in four groups, according to their pri- showed only low sequence identity to anti-fungal pep- mary structure, S–S bond pattern and biological func- tides as Rs-AFP1, a plant defensin from radish seeds tions. While ␥-thionins from types I and II are mostly (Raphanus sativus), Ah-AMP1 from horse chestnut known as toxic proteins to bacteria and fungi, pro- (Aesculus hippocastanum) and to drosomycin from P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253 2241 1-zeathionin, ␥ 1-purothionin, ␥ to glycines involved in structure -hordothionin, ␥ -thionin), Psd1. (B) Hellethionin, viscotoxin, ligatoxin B, phoratoxin, ␥ -hordothionin, Nt-thionin (flower-specific ␻ 1, ␣ 2-zeathionin, MsDef1, MtDef2, Pp-AMP1, Tu-AMP1, SI ␥ pseudo-thionin (Pth-St 1). Black arrows correspond to probable positive charged residues involved in biological activity, gray arrows correspond flexibility and asterisks correspond to conserved half-cystines. Fig. 1. Plant thionins primary structure analyses. Sequences are found at NCBI data bank. (A) Pp-thionin, crambin, Cp-thionin, 2242 P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253

D. melanogaster (Lay et al., 2003). These data corrobo- ture stability (Castro, Fontes, Morhy, & Bloch, 1996). rate to studies developed with Cp-thionin, a protein Furthermore, black arrows indicate conserved arginine from cowpea seeds (Vigna unguiculata), which rev- or lysine residues on ␥-thionins. In spite of this same ealed an enhanced identity of this ␥-thionin (57–81%) residue was not totally conserved in ␥-thionins, the con- with other protein classes, which include sulfur-rich struction of chimeras of M. sativa and M. truncatula proteins, proteinase and ␣-amylase inhibitors and ␥-thionins indicated that a positive charged residues at anti-fungal peptides. In fact, Cp-thionin was only able C-termini is critical for anti-microbial activity. Glycine to inhibit trypsin-like serine proteinases, while the residues, indicated by gray arrows, are highly con- other activities were not observed (Melo et al., 2002) served for ␥-thionins group, being important for pep- (Table 1), concluding that, for a while, it is impossible tides ﬂexibility and helping on function development to determine the function using primary structure. (Spelbrink et al., 2004). Analysis on phylogenetic tree Analyzing primary structures of different thionins, with thionins revealed that ␣ and ␤ could be grouped in its possible to observed that ␣- and ␤-thionins are more a separated clade (Fig. 1). Otherwise, ␥-thionins could similar between themselves than with ␥-thionins class be found for all tree clusters and anti-fungal ␥-thionin (Castagnaro, Marana, Carbonero, & Garc´ıa-Olmedo, tends to be grouped on left tree side, which could 1992, 1994). Last one have a smaller amino acid suggest primary structure conservation for this evolu- sequence, although Cp-thionin and crambin present tionary function (Fig. 2). Nevertheless, ␣-amylase and larger amino acid sequences (Jelsch et al., 2000; Melo proteinase inhibitors, as well anti-bacterial ␥-thionins et al., 2002). Cysteine residues are almost totally con- and peptides with mammalian cell death activity are served between ␥-thionins (indicated by an asterisk), distributed randomly into the tree, showing no direct as showed in Fig. 1A and B, being important to struc- evolutionary tendency.

Table 1 Source and function of most described thionins SwissProt code Protein Source Function Reference P83399 Cp-thionin Vigna unguiculata Proteinase inhibition Melo et al. (2002) P01542 Crambin Crambe abyssinica Sweet taste Teeter et al. (1981) P20230 ␥-Hordothionin Hordeum vulgareum ␣-Amylase inhibition and cell Mendez et al. (1990) death P32026 Flower-speciﬁc Nicotiana tabacum Putative pathogen defense Gu et al. (1992) gamma-thionin (Nt-thionin) 1NBLA Hellethionin Helleborus purpurascens Cell death Milbradt et al. (2003) AAG40321 MsDef1 Medicago sativa Anti-fungal activity Spelbrink et al. (2004) AAQ91287 MtDef2 Medicago truncatula Anti-fungal activity Spelbrink et al. (2004) – Pp-AMP1 and Pp-AMP2 Phyllostachys pubescens Anti-fungal activity Fujimura et al. (2005) Q8GTM0 NaD1 Nicotiana alata Anti-fungal activity, Lay et al. (2003) proteinase inhibition P20158 ␥1-Purothionin Triticum turgidum Insecticidal activity Colilla et al. (1990) P07504 Pp-thionin Pyrularia pubera Anti-bacterial activity, clotting Vernon, Evett, Zeikus, and system substitute Gray et al. (1985) S05594 Pseudo-thionin (Pth-St1) Solanum tuberosum Anti-bacterial activity Moreno et al. (1994) 7451437 SI␣1 Sorghum bicolor ␣-amylase inhibition, Bloch & Richardson (1991) anti-fungal activity – SPE10 Pachyrrhizus erosus Anti-fungal activity Song et al. (2005) – VaD1 Vigna angularis Anti-fungal and anti-bacterial Chen et al. (2005) activity P81008, P81009 ␥1-Zeathionin, ␥2-Zeathionin Zea mays Toxicity to animal cells Castro et al. (1996) – Fa-AMP1 and Fa-AMP2, Fagopyrum esculentum Anti-fungal and anti-bacterial Fujimura et al. (2003) activity – Tu-AMP 1 and Tu-AMP 2 Tulipa gesneriana Anti-fungal and anti-bacterial Fujimura et al. (2004) activity P81929 Psd1 Pisum sativum Anti-fungal activity Almeida et al. (2002) P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253 2243

Fig. 2. Evolutionary phylogenetic tree of plant thionins.

Differently of primary structure, which shows no structure of NaD1 is a common motif that consists evidential correspondence between function and high of a cysteine-stabilized ␣␤ (CS␣␤). Otherwise, ␣- sequence homology, tertiary structure of most ␥- helix shows an unusual structure, due the presence thionins are highly conserved in all functional groups of two residues (Pro18 and Pro19) at “trans” confor- (Almeida et al., 2003; Lay & Anderson, 2005; Mendez et al., 1996; Nitti et al., 1995). One ␣-helix and three anti-parallel ␤-sheets basically composed 3D structures of ␥-thionins, creating a typical amphipatic two- layer ␣␤ sandwich (Bruix et al., 1993, 1995)(Fig. 3). NaD1 and alfALP, showed thus a typical composition, where strands presented a ␤1a␤3a␤2 topology, indicating in a first view, a similar mechanism of action Fig. 3. Plant thionins three-dimensional structures comparison. 1BHP: ␤-purothionin (left); 1GPS: ␣1-purothionin (middle); 1 GPT: between both them. However, this is not particularly ␥2-hordothionin (right). 3D structures were drawn using SPDB- true for ␥-thionin family. The central features of 3D Viewer 3.7 (Guex & Peitish, 1997). 2244 P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253 mation, observed in structure N-termini (Gao et al., 2000; Nitti et al., 1995). ␥-Thionins 3D structures were also successfully compared to other proteins, such as scorpion neurotoxins, human endothelins, snake safarotoxins, opamins and animal defensins (Castro & Vernon, 1989, 2003; Li et al., 2002; Pineiro, Diaz, Rodriguez-Palenzuela, Titarenko, & Garc´ıa-Olmedo, 1995; Romagnoli et al., 2003). This correspondence is also genuine to SI␣1, a ␥-thionin from Sorghum bicolor seeds, which showed similar topology of scorpion toxins and charybdotoxins (Bloch et al., 1998). Hence, it was observed that only residues Ser7, Glu28, and Gly34 are fully conserved (Bloch et al., 1998). Fig. 4. Primary and tertiary structures comparisons of major (A) and Despite this protein class have low sequence homol- minor (B) forms of crambins. Black arrows and bold letters indicate ogy, it seems that ␥-thionins, scorpion neurotoxins differences at residues level. 3D structures were drawn using SPDB- and insect defensins evolved from a unique ancestor viewer 3.7 (PDB code 1AB1—minor form; 1CNR—major form) (Fig. 2). In addition to this group, some authors sug- (Guex & Peitish, 1997). gested that human endothelins, snake sarafotoxins and bee apamins might also have originated from one sin- two micro heterogeneous types based on primary struc- gular ancestor through exon shuffling. Nevertheless, in ture (Teeter et al., 1981), showing no differences in their these three protein classes, the CSH motif seems to tertiary structures (Jelsch et al., 2000; Teeter, Ma, Rao, have been excised and reintegrated in various orien- & Whitlow, 1990; Yamano & Teeter, 1994; Yamano tations during evolution, generating divergent protein et al., 1997). Furthermore, crambin Pro25 may pro- structures (Froy & Gurevitz, 1998). mote a small angulation in the protein backbone, cre- A deep analysis of ␥-thionins structures showed that ating small conformational differences between both small differences could be found. One clear example forms. If these comparisons were extended a little bit is observed in ␣-hordothionin isolated from barley ker- more, a very low sequence homology and a inhomoge- nels (H. vulgareum), an unusual ␥-thionin protein. Sev- neous distribution of basic amino acids along primary eral ␥-thionins structures descriptions (crambin and ␤- structure in crambins, ␥-thionins, ␣- and ␤-thionins hordothionin) demonstrated a similar protein-fold a the could be observed, directly reflecting in 3D struc- capital letter L, with two ␣-helices and a short stretch tures (Fant, Vranken, Broekaert, & Borremans, 1998). of anti-parallel ␤-sheet, connected by several turns or While crambin and ␣1-purothionin have letter T shape, bends of various lengths (Froy & Gurevitz, 1998; Oita, with the vertical axis composed by two anti-parallel Ohnishi-Kameyama, & Nagata, 2000). Nevertheless, at ␣-helices and extended strands in a horizontal arm, ␥-hordothionin was observed a protein fold-like simi- ␥1-purothionins and ␥1-hordothionins showed a letter lar to ␥-purothionin, which is composed by one ␣-helix L fold, with only one ␣-helix in parallel arrangement running in opposite direction to a short anti-parallel to three-stranded ␤-sheet (Bruix et al., 1993)(Fig. 3). beta-sheet strain (Castagnaro et al., 1994)(Fig. 3). Despite of differences and similarities of ␥-thionins Some differences were also observed on crambin, a structures, for this time is impossible to relate struc- hydrophobic thionin from C. abyssinica, which showed ture and function. Several efforts have been done in this two isoforms: a major form named PL and a minor form research field (Lay et al., 2003; Schaaper et al., 2001), determined SI (Yamano & Teeter, 1994). Differences but no proved relation was found (Almeida, Cabral, between both crambin amino acid sequences, is situ- Kurtenbach, & Zingali, 2000). It has been demon- ated on residues at position 22, which contains a serine strated that three-dimensional structure of ␥-thionins or a proline, while at position 25, a leucin or an isoleucin might not be related to their mechanisms of action, residue could be found (Jelsch et al., 2000; Spronk, but could be correlated to surface distribution of amino Linge, Hilbers, & Vuister, 2002; Teeter, Mazer, & acid residues (Almeida, Cabral, Kurtenbach, Almeida, L’Italien, 1981)(Fig. 4). Thus, crambin appears to have & Valente, 2002; Melo et al., 2002). The mechanism of P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253 2245 action of Psd1, a peptide isolated from Pisum sativum, calcium is chelated and pest ␣-amylases destabilized, was suggested by comparison with other ␥-thionins, leading to a remarkable enzyme inhibition. Hence, according to their tertiary structure and amino acid ␥-hordothionins inhibition activity occurs only against charges (Almeida et al., 2000). 3D structure similarities calcium-dependent ␣-amylases (Castro & Vernon, between Psd1 and other peptides did not revealed simi- 2003). It is also important to remind that ␣-amylases lar function, but a comparison between surface charges are not the unique enzyme class inactivated by ␥- distribution of several ␥-thionins leaded to some func- thionins. A peptide isolated from Cassia fistula seeds, tional similarities as anti-fungal, bactericidal and chan- with four disulfide bonds and structural similarities to nel blockers (Almeida et al., 2000, 2002). ␥-thionins group, showed inhibitory activity against Generally, plant ␥-thionins are composed by one serine proteases. This peptide was the first example of subunit, being found in monomeric forms (Almeida a ␥-thionins with activity against trypsin-like enzymes et al., 2002; Bloch et al., 1998). On the other hand, (Wijaya et al., 2000). Furthermore, a ␥-thionin from two plant ␥-thionins, one from Pachyrrhizus ero- cowpea seeds (Vigna unguiculata) also presented sus and other from V. unguiculata, showed the abil- inhibitory activity against several trypsins. Inhibitory ity to dimerism (Melo et al., 2002; Song et al., mechanism was suggested by molecular modeling, 2005). Although the real multimerization mechanism where catalytic site could be blocked by cowpea is unknown, a hypothesis was proposed by molecular ␥-thionin. Inhibitor residue Lys11 occupy a specific modeling analyses, suggesting that hydrophobic inter- pocket, resulting in a canonical fashion mechanism actions could be formed between both monomers (Song with a stoichiometry of interaction Cp-thionin–BPT et al., 2005). Discovery of novel dimeric defensins (bovine pancreatic trypsin) as 1:1 (Melo et al., 2002). could improve the mechanism of action understand- The association between Cp-thionin and trypsin might ing, since is suggested that those peptides could form be water-mediated, as like in the case of bovine pancre- dimers or multimers during ion-permeable pores for- atic trypsin inhibitor (BPTI) (Freitas, Ikemoto, & Ven- mation in target cells (Schibli et al., 2002; Song et al., tura, 1999; Huber et al., 1974). However, Cp-thionin 2005). was not capable to inhibit chymotrypsin, making Cp- thionin different from Bowman–Birk inhibitors, which can inhibit both trypsin and chymotrypsin (Melo et al., 3. Enzyme inhibition 2002). Furthermore, recent studies showed that NaD1 exhibited anti-fungal activity against plant pathogens In the last decade, an impressive discovery showed in vitro, as well toward insects as Helicoverpa that some ␥-thionins are able to inhibit digestive armigera and H. punctigera, by inhibiting trypsin-like enzymes, bringing ␥-thionins to a select group of and chymotrypsin-like enzymes (Lay et al., 2003). plant proteins synthesized in response to insect-pests As many other ␥-thionins, the structure of CS␣␤ (Franco, Rigden, Melo, & Grossi-de-Sa,´ 2002; Melo motif may be the key for NaD1functional activities. et al., 2002). Some ␥-thionins described until now However, despite of residues conservation, no concrete are capable to inhibit insect ␣-amylases (Bloch & fact was obtained about ␥-thionin enzyme selectivity. Richardson, 1991), and others could inhibit serine proteases (Melo et al., 2002; Wijaya, Neumann, Condron, Hughes, & Ploya, 2000). In this field, ␥-hordothionins 4. Anti-bacterial activity isolated from sorghum (S. bicolor) was the first example of a ␥-thionin able to inhibit insect ␣-amylases. ␥-Thionins are known for their effectiveness against This peptide inhibited ␣-amylases from cockroach bacteria (Table 1), showing this biological activ- Periplaneta americana and grasshopper Schistocerca ity with extreme specificity and frequently show- americana, but was incapable to inhibit mammalian ing no toxicity against other organisms (Iwai et al., ␣-amylases (Bloch & Richardson, 1991). It has 2002; Moreno, Segura, & Garc´ıa-Olmedo, 1994; Villa- also been speculated that the inhibitory activity of Perello´ et al., 2003). Furthermore, these proteins ␣-amylases has a peculiar mechanism of action. This appear to be specific to a certain group of bacte- ␥-thionin forms a Ca2+–SI␣1 complex, suggesting that ria. ␥-Thionins that are able to inhibit Gram-negative 2246 P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253 bacteria growth rarely decrease Gram-positive bac- meability modification (Titarenko, Lopez-Solanilla,´ teria growth, and the opposite is also valid (Diaz, Garc´ıa-Olmedo, & Rodr´ıguez-Palenzuela, 1997). Nev- Carmona, & Garc´ıa-Olmedo, 1992; Garc´ıa-Olmedo, ertheless, the involvement of a specific receptor has Molina, Alamillo, & Rodriguez-Palenzuela, 1998). not been ruled out, since ␥-thionins binds to specific Contrary to most anti-microbial ␥-thionins, a pep- phospholipids. ␥-Thionin interaction with membranes tide isolated from spinach (S. oleracea) is an impor- appears to be the first step in the exposure of toxic- tant exception for present lethal activity against both ity, followed by a cationic unbalance (Caaveiro et al., Gram-positive bacteria, such as Clavibacter michi- 1997). Otherwise, in plants, ␥-thionins are lead to acti- ganensis, and Gram-negative bacteria, as Ralstonia vate signaling networks that regulate innate responses, solanacearum. Furthermore, this peptide also demon- including plant hypersensitive reaction (Li et al., 2002). strated enhanced activity against phytopathogenic Another important question is related to specificity. fungi, such as F. culmorum, F. solani, Bipolaris may- Which are the residues involved in thionin–bacteria dis and Colletotrichum lagenarium (Segura, Moreno, interaction? How could we change the anti-bactericidal Molina, & Garc´ıa-Olmedo, 1998) indicating that activity to anti-fungal activity, using molecular biology spinach ␥-thionin showed a very large spectrum of techniques? Some thionins present a peculiarity that action. Similar results were obtained using two pep- differs from one another, having the capacity of being tides, Fa-AMP1 and Fa-AMP2, isolated from seeds toxic to microbes. Earlier studies have determined a of buckwheat (Fagopyrum esculentum), which also pseudo-thionin from S. tuberosum 1 (Pth-St1) that acts presented inhibition activity against Gram-negative only against bacteria, as potato pathogens C. michi- and positive bacteria (Fujimura, Minami, Watanabe, ganensis and P. solanacearum. Additionally, Pth-St1 & Tadera, 2003). Furthermore, two anti-microbial ␥- showed no enzyme inhibitory activity and did not affect thionins isolated from bulbs of tulip (Tulipa gesneriana cell-free protein synthesis (Moreno et al., 1994). A2 ␥- L.) showed activity against bacteria, such as Erwinia thionin from P. pubera (Pp-TH), for example, showed carotovora, Agribacterum radiobacter, Agrobacterium in vitro anti-bacterial activity against several repre- rhizogenes, Clavibacter michiganensis and Curtobac- sentative pathogens, such as Gram-negative bacteria terium flaccumfaciens (Fujimura, Ideguchi, Minami, R. melioti and X. campestris, and also against Gram- Watanabe, & Tadera, 2004). Recently, two peptides positive bacteria C. michiganensis (Bung, Wolters, from Japanese bamboo shoots, Pp-AMP 1 and Pp- & Apel, 1992). This peptide contains one aspartic AMP 2, demonstrated activity against plant pathogenic residue instead of arginine at position 32, commonly bacteria, such as E. carotovora, A. radiobacter, A. found in several other ␥-thionins. The presence of rhizogenes, C. michiganensis and C. flaccumfaciens Asp32 showed, a significant importance in antibiotic (Fujimura, Ideguchi, Minami, Watanabe, & Tadera, activity against diverse Gram-negative bacteria, and 2005). retained the same activity against innumerous fungi As described before, ␥-thionins exhibit a very con- species as F. oxysporum, P. cucumerina and B. cinerea served structure (Fig. 3). This amphypatic feature is (Villa-Perello´ et al., 2003). It is important to under- closely related to disruption of microbial membranes stand that, in spite of severe efforts, the real mech- and phospholipid lypossoma. The anti-bacterial molec- anism of ␥-thionin specificity remains unclear and ular mechanism of defense remains unclear and not obscure and the discovery of these factors will be completely elucidate, but some mechanisms have been a remarkable goal on development of new specific suggested. It has been known that bacterial liphos- antibiotics. pho surface (LPS) is a permeable barrier that confers resistance to anti-microbial agents and environmental modifications resulting in an increasing of sensitiv- 5. Anti-fungal activity ity to hydrophobic and cationic compounds (Nikaido, 1989). One hypothesis for the role of ␥-thionins on Anti-fungal ␥-thionins are more numerous, when anti-bacterial activity is inferred on the way that posi- compared to bactericidal ␥-thionins reported until tively charged proteins interact with negatively charged 2005 (Milbradt, Kerek, Moroder, & Renner, 2003; membrane phospholipids, following a membrane per- Selitrennikoff, 2001; Vigers, Roberts, & Selitrennikoff, P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253 2247

1991). However, some anti-fungal ␥-thionins also on sphingolipids biosynthesis, IPT1 and SKN1, leaded present toxicity to other organisms (Berrocal-Lobo to an increase on susceptibility to ␥-thionin DmAMP1 et al., 2002; Diaz et al., 1992; Vigers et al., 1991). (Thevissen et al., 2005). One example is observed in peptides isolated from Another question not well answered is how the bulbs of tulip and buckwheat, already described before pores are maintained open into fungal cell membrane (Fujimura et al., 2003, 2004). These peptides showed is done by repulsion of ␥-thionins positive charges or activity both against bacteria and fungi (Fusarium by their hydrophobic interaction with phospholipids oxysporum and Geotrichum candidum), although their bilayer. Consequently, if the positive charges of ␥- anti-fungal activity is higher than anti-bacterial activity thionins maintain an open pore, ions potassium and/or (Fujimura et al., 2004). calcium will be repulsed to cell outside, as observed in Anti-fungal mechanism of action seems to be occa- the scheme of proposed mechanism of action (Fig. 5). sioned by an interaction to a specific receptor as an ion The attraction of ␥-thionins to fungi membrane recep- channel or a sphingolipid (Bloch et al., 1998; Florack tors could also be made by ionic interaction between & Stiekema, 1994; Kushmerick, Castro, Cruz, Bloch, ␥-thionin side chain residues and charged glycolipids & Beirao,˜ 1998; Thevissen et al., 1996, 2005). An inter- from cell surface (Almeida et al., 2002; Selitrennikoff, esting observation was that electrostatic interaction ␥- 2001). thionins-cell membrane could be reduced by divalent As bactericidal ␥-thionins, some of them also show cations, as Ca2+ (Lay et al., 2003). ␣-Hordothionins certain peculiarities that make them extremely pow- from barley are capable of interacting electrically with erful toward fungi. SI␣1, for example, is a protein fungal lipid bilayer, linking to membrane surface (but from S. bicolor that exhibits a dual affectivity toward not insert into it), leading to permeabilization and dis- bacterial and fungi (Bloch et al., 1998). It shows a rupting the membrane organization. Consequently, the typical ␣ + ␤ sandwich structure with a helix held by fungicide mechanism may not occur through a direct two disulfide bridges localized against the sheet and protein–protein interaction, but via lipid membrane their mechanism of action is still under debate, seems receptor (Thevissen et al., 1996). that ␥-thionins free loops conformations are impor- How defensins causes severe membrane damages tant factors for anti-microbial activity (Bloch et al., after interaction with surface fungal cell is a real mys- 1998; Selitrennikoff, 2001). By the fact that in SI␣1, tery and continues poorly understood. One possibility the first loop that connects ␣-helix and the second was described recently where ␥-thionins might bind strand is longer than the same loop observed in ␥- to glycolipids at fungal membrane surface (Ferket, and ␻-thionins, the second loop, positioned between Levery, Park, Cammue, & Thevissen, 2003; Thevissen, second and third strand, is shorter in SI␣1 when com- Ferket, Francois, & Cammue, 2003; Thevissen et al., pared to both thionins. For these reasons, SI␣1 anti- 2004; Thomma et al., 2003a,b). In this case, glycol- microbial activity is probably less effective than ␥- and ipids will work as membrane receptors, despite two ␻-thionins, although it is still considered a powerful or more proteins could also be involved (Thevissen tool for pathogen growth inhibition (Bloch et al., 1998). et al., 2003, 2004, 2005) and consequently a pore will Other purothionins also demonstrate a wide range of be formed, leading to ion influx/efflux. This mecha- anti-fungal activity. ␤-Purothionin purified from wheat nism, which blocked ion Ca2+ influx through fungi cell, showed to be capable to kill both fungi and mam- was observed (Spelbrink et al., 2004). mal cells by forming monovalent cation-selective ion Glucosylceramides and sphingolipids are frequently channels in cell membrane, affecting osmotic perme- described as cell membrane receptors for ␥-thionins. It ability of fungal cells (Hughes, Dennis, Whitecross, was demonstrated that glycolipids knockout increase Liewelly, & Gage, 2000). Purothionins have also pre- fungi resistance against ␥-thionins deleterious effects sented lytic activity against R. solani, alone or in asso- (Ferket et al., 2003; Thevissen et al., 2003, 2004, ciation with other anti-fungal proteins (Oard, Rush, & 2005). Otherwise, it was observed that fungi suscepti- Oard, 2004). Cell disruption increases the possibility bility might be related to specific sphingolipid manno- of lyses, and consequently, enhances cell death. ␣1-, syl diinositolphosphoryl ceramide biosynthesis. Yeast ␣2-Purothionins and ␣1-hordothionin also appear to mutants revealed that expression of two genes involved form ion channels in artificial bilayers and biological 2248 P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253

Fig. 5. Probable ␥-thionins mechanism of action, by using glucosylceramides as receptors for fungi cell membrane insertion. (A) Glucosylce- ramide molecules situated on cell membrane; ␥-thionins at free form; (B) interaction between ␥-thionins and glucosylceramide; (C) repulsion of ␥-thionin into cell membrane by their positive charges, leading to membrane disruption; (D) membrane destabilization and ion efflux (K+). ( ) ␥-Thionin; ( ) glycosylceramide molecule; ( ) fungi cell bilayer; K+—ion potassium. (I) Correspond to cell inside and (O) to cell outside. membranes, promoting similar cell damage in fungi 1992; Milbradt et al., 2003; van der Heuvel, Hulzink, (Hughes et al., 2000). Likewise, a peptide isolated from Barendse, & Wullems, 2001; Xu et al., 2001). Fur- barley grain (BCP-2) appears to have great similar- thermore, an endogenous gene could also be dupli- ity to barley ␣-thionin, having anti-microbial activity, cated and re-introduced into the same plant genome, especially against fungi. BCP-2 bounds to constitutive in order to improve the resistance, decreasing the polysaccharides, such as glucosylceramides and sph- necessity of enhanced quantities of pesticide used on ingolipids, from fungal cell walls (Kushmerick et al., agriculture. 1998; Oita et al., 2000), but these interactions are not ␥-Thionin genes from oat (Avena sativa) were very well understood. Nevertheless, authors suggested transferred to rice (Oryza sativa), producing trans- that it may increase the fungi cell wall sensibility to genic plants resistant to bacteria, such as Burkholde- environment hostilities, leading to cell perturbations, ria plantarii and B. Glumaeby (Segura et al., 1998). and consequently, fungal cell lyses (Selitrennikoff, It was also demonstrated that barley ␣-hordothionin 2001). expressed in transgenic tobacco leaded to an enhance of resistance to bacterial pathogens like P. syringae and also fungus F. oxysporum when over expressed 6. Biotechnological issues into leaves of A. thaliana (Carmona, Molina, Fernan- dez, Lopez-Fando, & Garc´ıa-Olmedo, 1993; Epple, ␥-Thionins are important tools for genetic improve- Apel, & Bohlmann, 1997; Hughes et al., 2000). Plants ment and development of transgenic plants resistant to could also develop resistance against pathogens dur- certain pathogens. Transgenic plants expressing higher ing cold weather through induction of defense pro- levels of ␥-thionins from other plants could increase the teins. A gene from wheat – Tad1 – that encodes pathogenic resistance, reducing crop losses (Bohlmann a ␥-thionin (TAD1) seems to be involved in resis- et al., 1998; Gu, Kawata, Morse, Wu, & Cheung, tance induced by low temperatures against pathogens P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253 2249 during could weather. TAD1 also showed activity ficity, ␥-thionins probably might reduce the collateral against plant bacteria P. cichorii, evidencing their effects caused by common remedies used against these activity toward biotic and abiotic stresses (Koike, illnesses. Okamoto, Tsuda, & Imai, 2002). Moreover, it was also observed that expression of ␣1-hordothionin from endogenous leaves of A. thaliana results in resis- 7. Conclusions tance increasing against F. oxysporum (Hughes et al., 2000). ␥-Thionins appeared to have very strong sequence Moreover, it has been isolated ␥-thionins gene with similarities between one another, as well a struc- toxicity to fungi (Epple, Apel, & Bohlmann, 1995; tural identity composed by the organization of ␣- Epple et al., 1997). Two of them, from A. thaliana, helices and ␤-sheets. In contrary of few thionins, codify pre-proteins of ␥-thionins super family, and such as ␣-hordothionin, most ␥-thionins seems to are designed as Thi2.1 and Thi2.2. Transcript gene present similar secondary and tertiary structure. More- of Thi2.1 was found in rosette leaves and flowers. over, they demonstrated some identity to other pro- The expression of Thi2.1 gene appeared to be highly tein classes, as insect defensins and scorpion tox- inducible by pathogens, silver nitrate and methyl- ins. This homology between ␥-thionins suggests high jasmonate, indicating that this gene may be related conservation during evolution, which might confirm to plant defense against pathogens and it probably their important role on plant defense against exter- may be induced by a signal transduction pathway nal pathogens. Besides, different members of plant (Epple et al., 1995). Thi2.1 also enhanced resistance ␥-thionin family showed structure and sequence simi- from A. thaliana plants against neucrotrophic fungi larity, no structure–function relation could be observed. by an octadecanoid pathway (Vignutelli, Wasternack, It seems that toxicity requires an electrostatic inter- Apel, & Bohlmann, 1998). Furthermore, Thi2.1 was action of positively charged ␥-thionins with certain induced by phytophatogenic fungi presence increasing lipid domains. Theses domains could be composed for the resistance of ecotype Columbia (Col-2) against the glycolipids, which mediate signal transduction from attack of F. oxysporum (Epple et al., 1995). As plant environment to eukaryote organisms. Interaction of ␥-thionins have demonstrated anti-microbial activities ␥-thionins to fungi membrane surface by certain recep- and is being target as a fresh instrument to development tors can elucidate mechanism of action. In this field, novel antibiotic, isolation and experimental character- glycosylceramides and sphingolipids, located on cell ization of these proteins could provide specific drugs surface, have demonstrated the capability of interac- to human pathogens (Thomma et al., 2003a,b). The tion to ␥-thionins and an important target to fungi cell utilization of ␥-thionins might focuses on cure of sev- permeabilization (Ferket et al., 2003; Thevissen et al., eral bacterial diseases that affect immune-depressed 2004). These peptides can form pores, but they neither patients, such as pulmonary and nosocomial infec- present redox activity nor activate secondary messen- tions. However, fungi medicines will also be produced ger enzymes (Hughes et al., 2000). Their toxicity to to combat dermatophytosis, which are the most com- plant pathogens in vitro, such as bacteria and fungi mon fungal infection developed in humans (Cowan, may reflect a direct role in plant defense (Florack & 1999). From all these uses, ␥-thionins still present Stiekema, 1994). However, the binding to a protein another important function in medical care (Bussing, receptor could not be discarded. Schaller, & Pfuller, 1998; Bussing et al., 1999; Johans- There are still lots to be discovered about ␥-thionin son et al., 2003). As some thionins are capable to super family, especially related to their mechanism of inhibit mammal cells growth, they are now been stud- action. However, all findings already done provided ied for a future development of anti-carcinogenic drugs important information for development of novel antibi- (Bussing et al., 1998; Johansson et al., 2003; Li et otics, and either a different strategy against aggressive al., 1999, 2002). With well-selected and powerful pep- tumor cells. Biotechnology has already being very use- tides, the introduction of a medicine developed from ful on cultivars in agriculture, but still needs novel tools ␥-thionins could become a novel selective treatment for genetic improvement on increasing plant resistance way for the cure of several cancers. Due their speci- against pathogens. 2250 P.B. Pelegrini, O.L. Franco / The International Journal of Biochemistry & Cell Biology 37 (2005) 2239–2253

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