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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 An´alises Proteˆomicas e Bioqu´ımicas, P´os-Gradua¸c˜ao em Ciˆencias Genˆomicas e Biotecnologia, Universidade Cat´olica de Bras´ılia, SGAN Quadra 916, M´odulo 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 Fig. 1. Plant thionins primary structure analyses. Sequences are found at NCBI data bank. (A) Pp-thionin, crambin, Cp-thionin, ␥-hordothionin, ␥1-purothionin, ␥1-zeathionin, ␥2-zeathionin, MsDef1, MtDef2, Pp-AMP1, Tu-AMP1, SI␣1, ␻-hordothionin, Nt-thionin (flower-specific ␥-thionin), Psd1. (B) Hellethionin, viscotoxin, ligatoxin B, phoratoxin, pseudo-thionin (Pth-St 1). Black arrows correspond to probable positive charged residues involved in biological activity, gray arrows correspond to glycines involved in structure flexibility and asterisks correspond to conserved half-cystines. 2241 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
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