From plant physiology to pharmacology: fusicoccin leaves the leaves
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This review highlights 50 years of research on the fungal diterpene fusicoccin, during which the molecule went from a tool in plant physiology research to a pharmacological agent in treating animal diseases.
Fusicoccin is a phytotoxic glycosylated diterpene produced by the fungus Phomopsis amygdali, a pathogen of almond and peach plants. Widespread interest in this molecule started when it was discovered that it is capable of causing stomate opening in all higher plants, thereby inducing wilting of leaves. Thereafter, FC became, and still is, a tool in plant physiology, due to its ability to influence a number of fundamental processes, which are dependent on the activation of the plasma membrane H+-ATPase. Molecular studies carried out in the last 20 years clarified details of the mechanism of proton pump stimulation, which involves the fusicoccin-mediated irreversible stabilization of the complex between the H+-ATPase and activatory 14-3-3 proteins. More recently, FC has been shown to influence cellular processes involving 14-3-3 binding to client proteins both in plants and animals. In this review, we report the milestones achieved in more than 50 years of research in plants and highlight recent advances in animals that have allowed this diterpene to be used as a 14-3-3 targeted drug.
KeywordsDiterpene phytotoxin Plasma membrane H+-ATPase, 14-3-3 proteins Protein–protein interaction Drug design
Milestones of FC research
First studies on the phytotoxic activity of P. amygdali
Purification of FC from coltures of P. amygdali
Ballio et al. (1964)
FC opens stomata
Turner and Graniti (1969)
Detection of FC binding sites in microsomal fractions of maize coleoptiles
Dohrmann et al. (1977)
Model for FC stimulation of the plasma membrane H+-ATPase
Identification of 14-3-3 proteins in FC receptor preparations
FC induces the association of 14-3-3 proteins with the H+-ATPase
The H+-ATPase/14-3-3 complex generates the FC receptor
FC affects amphibian embryogenesis
Bunney et al. (2003)
Crystal structure of the FC/14-3-3/H+-ATPase peptide ternary complex
Würtele et al. (2003)
FC induces platelet aggregation
Camoni et al. (2011)
FC stimulates axon growth and regeneration
Discovery and early studies
The interest in FC prompted efforts to obtain large amounts of the toxin which were actually produced by pilot-scale (3000 L) cultures of the fungus (Ballio et al. 1968b), to allow extensive investigations of its effects in plants. These studies led to the discovery of other physiological effects elicited by FC including modification of tissue growth, opening of stomata, nutrient uptake, and breaking of seed dormancy (Marrè 1979). At cellular level, growth is accompanied by H+ extrusion, hyperpolarization of the plasma membrane potential, and K+ uptake, an effect strikingly similar to that induced by the natural plant hormone auxin. From these circumstantial pieces of evidence, Marrè (1979) proposed that the basis of FC action could be the activation of a plasma membrane electrogenic proton pump, a model confirmed by experimental work in the successive years. Following progress in the purification of plasma membranes, a detailed biochemical characterization of the FC-induced stimulation of the H+-ATPase was carried out in two-phase partitioned plasma membrane vesicles. FC brings about an increase of the Vmax of the enzyme, together with a shift in its pH optimum (De Michelis et al. 1991; Lanfermeijer and Prins 1994). The conclusive demonstration that the H+-ATPase is the target of the toxin was obtained by expression of the H+-ATPase C terminus in yeast or complementation of the yeast H+-ATPase with the Arabidopsis thaliana AHA2 and Nicotiana plumbaginifolia PMA2 isoforms, which generated high affinity FC binding sites (Jahn et al. 1997; Baunsgaard et al. 1998; Piotrowski et al. 1998).
The synthesis of a radioactive and biologically active FC derivative allowed to start studies to identify FC targets in the plant cell. FC binding sites were detected for the first time in microsomal fractions of corn coleoptiles (Dohrmann et al. 1977), while it was subsequently ascertained that they occur in the plasma membrane of all plants, from liverworts to angiosperms (Meyer et al. 1993). The breakthrough in the search for FC receptors occurred in the nineties, when new advanced chromatographic techniques independently allowed three groups to identify members of the 14-3-3 protein family in FC receptor-enriched fractions from different plants (Marra et al. 1994; Korthout and de Boer 1994; Oecking et al. 1994). 14-3-3 proteins are ubiquitous regulators occurring as numerous isoforms in eukaryotic organisms (Aitken et al. 1992). However, at that time they were poorly characterized in animals and were virtually unknown in plants. Over successive years, an impressive body of accumulating evidence proved that these proteins are involved in the control of a number of pivotal physiological processes, such as cell cycle progression, apoptosis, cellular trafficking, and gene transcription (Fu et al. 2000; Hermeking 2003) and that in plants, they participate in further peculiar functions, such as regulation of nitrogen and carbon metabolism (Huber et al. 2002), ion transport (Aducci et al. 2002; de Boer 2002; Camoni et al. 2018) and hormone and light signaling (Taoka et al. 2011; Camoni et al. 2018).
14-3-3 proteins exert their effects by binding to phosphorylated client proteins, thereby modulating their sub-cellular localization, enzymatic activity, turn over, or their ability to associate with other proteins (Fu et al. 2000; Hermeking 2003). The structure of 14-3-3 proteins from various organisms has been clarified by X-ray crystallography (Xiao et al. 1995; Liu et al. 1995; Oecking and Jaspert 2009). Each monomer comprises nine anti-parallel α-helices and binds through its N-terminus a second monomer to assemble the functional dimeric protein. The 14-3-3 dimer has a characteristic W-like shape, with a conserved internal surface and a more variable external surface. The internal surface contains a conserved amphipathic cavity, which is responsible for the interaction with the phosphorylated target (Yaffe et al. 1997; Ottmann et al. 2007; Taoka et al. 2011). Analysis of the phosphorylated binding sequences of 14-3-3 clients revealed that 14-3-3 proteins recognize pSer (p = phosphate) and pThr-containing motifs called mode I (RSX(pS/pT)XP and mode II (RXY/FX(pS/pT)XP, respectively (Yaffe et al. 1997).
The plant FC receptor
The identification of the 14-3-3 binding site of the proton pump represented the first demonstration of the occurrence of a C-terminal binding motif for the interactions between 14-3-3 proteins and their clients. In fact, successive studies clarified that, whereas the majority of clients bind to 14-3-3 proteins through mode I and II motifs, a limited number of targets bind through C-terminal sequences, that were later defined mode III motifs ((pS/pT)X1-2-COOH, Coblitz et al. 2006). The resolution of the crystal structure of the ternary complex between FC, 14-3-3 proteins and the phosphorylated C-terminal peptide of the H+-ATPase shed light on the molecular basis of toxin binding (Würtele et al. 2003; Fig. 2b). The phosphopeptide is accommodated by the amphipathic groove that 14-3-3 proteins typically use to bind their targets, while FC is arranged next to the C terminus of the phosphopeptide, making contact both with the peptide and the 14-3-3 groove. Isothermal titration calorimetry (Würtele et al. 2003) and surface plasmon resonance (Fuglsang et al. 1999) experiments clarified that FC binds 14-3-3 proteins with very low affinity, whereas the peptide and the toxin reciprocally enhance, by approximately two orders of magnitude, their respective binding affinities, bringing about a strong stabilization of the ternary complex.
The search for further FC receptors in animals and plants
The occurrence of FC binding sites was initially reported to be restricted to higher plants, whereas they were not found in prokaryotes, fungi, algae, and animals (Meyer et al. 1993). Nevertheless, the identification of the FC receptor as a complex between 14-3-3 proteins and the H+-ATPase client raised the question as to whether other receptors exist, since many 14-3-3 protein targets have been described. The resolution of the structure of the ternary complex between FC, 14-3-3 proteins and the H+-ATPase provided the rationale to clarify that the stabilizing effect of FC occurs only with mode III clients, whereas the toxin is ineffective with mode I and II targets, which account for the vast majority of 14-3-3-interacting proteins (Camoni et al. 2013; Paiardini et al. 2014). In fact, in the latter interactions, FC does not fit into the 14-3-3 binding groove since it is usually occupied by a Pro residue in position +2 with respect to the pSer present in the mode I or II peptides. Nevertheless, even though to a reduced extent as compared to mode I and II clients, different mode III 14-3-3 clients have so far been identified in plants and animals, a fact that leaves open the question of the occurrence of further FC receptors and prompted studies to search novel FC targets. Bunney et al. (2003) reported that FC induced heterotaxia during the early development of Xenopus laevis embryos. Accordingly, the authors also showed the occurrence of cytoplasmic binding sites for [3H]FC with affinity comparable to that of plant receptors.
Since it has been well-documented that FC-related terpenoids possess anti-cancer properties (Honma and Akimoto 2007; Bury et al. 2013a, b; Rodolfo et al. 2016), and it is known that 14-3-3 proteins are implicated in the progression of various types of cancer (Hermeking 2003; Freeman and Morrison 2011), different studies have been performed to test whether FC can affect tumor cells proliferation or viability. It has been found that FC can induce apoptosis in various tumor cell types after priming with Interferon-α (de Vries-van Leeuwen et al. 2010). FC also inhibits the proliferation of glioblastoma cells (Bury et al. 2013a, b). In this system, FC is able to lower the cell growth rate by increasing the duration of cell division. Although the molecular mechanism which underlies the cytostatic effect is not known, it has been observed that the toxin decreases the in vitro activity of several protein kinases involved in cell cycle progression.
A further piece of evidence that FC can be a general regulator of the interactions between 14-3-3 proteins and mode III targets has been obtained in human platelets, where it has been demonstrated that the toxin stimulates the 14-3-3 association to the glycoprotein Ibα (Camoni et al. 2011). This protein is part of GPIb-IX-V, a complex that mediates the initial adhesion of circulating platelets to the sub-endothelial von Willebrand Factor. This, in turn, results in platelet activation and consequent aggregation. A similar mechanism involving FC stabilization of the binding of 14-3-3 proteins to a mode III client has been discovered in breast cancer cells (de Vries-van Leeuwen et al. 2013), where the activity of the estrogen receptor α (ERα) is negatively controlled by 14-3-3 proteins, which inhibit its dimerization. The FC-mediated stabilization of the ERα/14-3-3 complex determines the inhibition of the receptor activity, thereby hampering ERα-mediated gene activation and cell growth. In this case, the molecular basis of the FC interaction has been clarified by the crystallographic resolution of the ternary complex structure, which strictly resembles that of the complex between the 14-3-3 proteins and the plasma membrane H+-ATPase.
A different approach used to identify mode III FC receptors was based on a systematic in vitro analysis of the effect of FC on the interaction between 14-3-3 proteins and phosphopeptides with known human mode III binding sequences (Paiardini et al. 2014). This study established the structural requisites of mode III motifs that allow FC binding and the formation of the ternary complex. Isothermal titration calorimetry (ITC) and bioinformatics analysis revealed that the FC effect is solely dependent on the physiochemical properties of the residue in position + 1 with respect to the pSer/pThr. In general, the interaction is favored with hydrophobic side chains of the C-terminal amino acid. However, steric hindrance and/or limited plasticity of cyclic rings can obstruct the accommodation of FC in the 14-3-3-binding cavity, thereby hampering the assembly of the ternary complex (Paiardini et al. 2014).
A notable exception is represented by the FC effect on the interaction between 14-3-3 proteins and the cystic fibrosis transmembrane conductance regulator (CFTR), the anion and bicarbonate transporter implicated in cystic fibrosis (Stevers et al. 2016). In fact, it has been shown that FC is able to stabilize the interaction of 14-3-3 protein with a non-canonical binding motif of CFTR (RIpS253VIS), which does not resemble either the mode I/mode II or the C-terminal mode III binding motif. In this case, the crystal structure of the complex revealed that 14-3-3 and the CFTR phosphopeptide form a hydrophobic pocket that accommodates FC. The ternary complex is stabilized by hydrophobic interactions between the toxin and Val and Ile in position + 1 and + 2 to the pSer, respectively. FC-mediated stabilization of the interaction between 14-3-3 and CFTR increases transporter trafficking to the plasma membrane and hence chloride transport over the plasma membrane. The druggability by FC of the 14-3-3/CFTR complex may therefore be exploitable for a new approach for cystic fibrosis therapeutics.
14-3-3 proteins are highly expressed in the central nervous system (CNS), where they act as key mediators of nervous system development and axon guidance (Kent et al. 2010; Yam et al. 2012). Since CNS axons have a poor ability to regenerate, a fact that underlies persistent disability in spinal cord and nerve injuries, therapies that induce axon re-growth through drug-targeting of 14-3-3 could be a promising therapeutic approach. Very recently, Kaplan et al. (2017a) demonstrated that FC stimulates damaged axon growth in vitro and regeneration in vivo of rat cortical embryonic neurons and that the effect is mediated by 14-3-3 proteins. Purification of toxin-treated cell extracts by affinity chromatography with FC allowed identification of proteins containing putative mode III binding motifs. One of the identified proteins, the stress response regulator GCN1, has been shown to form a complex with 14-3-3 proteins which is stabilized by FC, thereby resulting in GCN1 degradation and neurite outgrowth and regeneration. This finding suggests that FC may be a promising drug to target 14-3-3 protein–protein interactions for CNS disease therapeutics (Kaplan et al. 2017b).
The search for further FC-sensitive 14-3-3 targets has been successfully accomplished also in plants where it has led to the identification of the A. thaliana K+ outward channel1 (KAT1) as a novel FC receptor (Saponaro et al. 2017). In fact, it has been demonstrated that 14-3-3 proteins associate to KAT1 by binding to a C-terminal site and that FC greatly stabilizes the interaction, thereby reinforcing the observed stimulatory effect of 14-3-3 proteins. Interestingly, the crystal structure of the ternary complex has revealed that FC is forced to assume an unusual conformation to fit the task owing to the steric hindrance of the C-terminal Asn within the mode III binding site (YFSpSN-COOH). It is worth noting that the stimulatory effect of FC on KAT1 provides a molecular rationale to previously unexplained patch-clamps data that showed that the toxin can alter K+ fluxes independently from the H+-ATPase-generated membrane potential (Blatt and Clint 1989).
Cotylenin and semisynthetic FC derivatives
The implication of 14-3-3 proteins in the control of fundamental cell processes, such as cell division, development and apoptosis, whose misfunctioning can lead to cancer and other diseases, has stimulated a great interest in the development of FC derivatives for therapeutic application (Milroy et al. 2013; Giordanetto et al. 2014).
Cotylenin A (CN, Fig. 1) is a diterpene glucoside closely related to FC which displays in vivo activities both in plants and animals similar to that of FC via the same mode of action (Honma 2002; Ottmann et al. 2009). However, the elucidation of the ternary complex between CN, 14-3-3 and prototype mode I peptides revealed that CN can also stabilize interactions with mode I and II motifs (Ottmann et al. 2009; Molzan et al. 2013). This peculiarity as compared to FC, due to the lack of C12 hydroxylation in CN, may underlie the differences in the activity of FC and CN in cancer cells (Ottmann et al. 2009; Milroy et al. 2013).
Nevertheless, it must be pointed out that FC is not completely specific for mode III motifs. In fact, even though the toxin does not stabilize the interaction of 14-3-3 with canonical mode I and II motifs, it can stabilize the 14-3-3 interaction with non-canonical internal motifs, which lacks the Pro residue in position + 2 (Stevers et al. 2016). To produce a selective molecule for mode III targets, a FC derivative (FC-THF) has been synthesized by introducing a tetrahydrofuran ring at C12 of the diterpene, to induce steric repulsion with any residue in position + 2. FC-THF specifically increases the association of 14-3-3 to the mode III target K+ channel TASK-3 without altering the interaction with canonical mode I and II clients (Anders et al. 2013).
An approach based on molecule dynamics has been used to rationally design semisynthetic FC derivatives with improved ability to stabilize the 14-3-3/client interaction (Ottmann et al. 2018). In these compounds, substitution of the 19-acetoxy group with an acetamide (FC-NAc) provided a hydrogen bond with an Asp residue present in the 14-3-3 groove, thus stabilizing the FC/14-3-3 interaction. Interestingly, FC-NAc showed increased activity in the INF-α-dependent growth inhibition of human ovarian carcinoma cells (OVCAR-3), demonstrating that structure-based modifications of FC can be a promising approach to enhance its therapeutic potency.
Moreover, since some cancer types are associated to increased 14-3-3 expression (Freeman and Morrison 2011), drug-based inhibition of 14-3-3 interaction with client proteins could be a convenient alternative strategy. This approach has been exploited to synthesize, within the cell, FC-peptide conjugates able to bind the 14-3-3 groove and thereby hampering their ability to interact with client proteins. (Maki et al. 2013; Parvatkar et al. 2015).
Conclusion and perspectives
FC was first recognized as a wilt-inducing phytotoxin able to irreversibly open stomata of higher plants. Intensive biological and biochemical studies have elucidated its value as a tool in studying plant physiology, since the toxin binds to a protein complex formed by the master enzyme of plant ion transport, the plasma membrane H+-ATPase and regulatory 14-3-3 proteins. X-ray crystallographic studies clarified the molecular details of this very peculiar interaction: FC specifically accommodates into a pocket at the interface between the H+-ATPase and a 14-3-3 protein; this pocket is generated by interaction of a C-terminal sequence on the proton pump and a unique 14-3-3 consensus sequence. This is known as mode III binding and is a much less common than canonical mode I and II consensus motifs. Why plants possess such a unique recognition mechanism for FC, a molecule that most plants never encounter, is unclear. One intriguing possibility is that the fungal product mimics endogenous compounds of similar structure and with similar regulatory functions (de Boer and de Vries-van Leeuwen 2012), as already described for other fungal metabolites like gibberellins. However, even though the occurrence of members of the fusicoccanes terpenoid family have been reported in fungi, bacteria, and plants, up to now no conclusive results have been obtained on the occurrence of FC-like compounds in plants (de Boer and de Vries-van Leeuwen 2012).
It is also conceivable that the capability of FC to bind to and deregulate the H+-ATPase was acquired successively, in the course of the evolutionary arms race between pathogens and plants, whereas initially the molecule accomplished less specialized functions typical of necrotrophic pathogenesis, such as membrane-disrupting activity. In this respect, it is worth noting that FC is an amphipathic molecule with detergent-like properties, able to desegregate liposomes at µM concentration (personal communication).
The second question raised from recent research is whether FC will be also a tool in pharmacological research, since both plants and animals possess a number of potential mode III 14-3-3 clients that are known to be involved in a wide array of physiological and/or pathological processes. In fact, whereas the development of small-molecule inhibitors of 14-3-3/client interaction is a relevant issue in pharmacology (Milroy et al. 2013; Giordanetto et al. 2014), in alternative cases drug-mediated stabilization of 14-3-3/client interaction may be a more convenient approach (Milroy et al. 2013; Giordanetto et al. 2014). In this respect, the peculiar nature of FC as a cell-permeable stabilizer of 14-3-3 interactions with mode III targets certainly represents a good starting point for research aimed to deepen the knowledge about the biochemical consequences of protein–protein interaction modulation, as well as to develop new therapeutic agents derived from FC.
Author contribution statement
All authors designed the outline of the article. LC and MM wrote the article. LC prepared the figures. SV and PA provided scientific feedback, critical comments and revised the article. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interests.
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