Genetic evolution of seed peptides

Sunflowers make ultrastable peptides by the proteolytic maturation of some unusual bifunctional seed storage proteins. This area of research asks: when did daisies start making ultrastable peptides and how are they evolving?

While pursuing the genetic origin of the small cyclic peptide SFTI-1 (Sunflower Trypsin Inhibitor-1), we stumbled upon an extraordinary case of protein hijack (Nature Chem Biol 2011). It transpired that the sequence for SFTI-1 was buried inside a napin-type seed storage albumin precursor we named PawS1 for Preproalbumin with SFTI-1. Using mass spectrometry we demonstrated the peptides were more widespread, found in other Helianthus species as well as Tithonia and even guayule (Parthenium argentatum). This suggests that the genetic event that created these ultrastable peptides is shared by an ancestral species that gave rise to these different genera.

We recently revealed a new class of peptides we called the PawS-Derived Peptides (PDPs) using a combination of heterologous PCR and de novo transcriptomics supported by mass spectrometry. This peptide family is at least 18 million years old (Plant Cell 2014).

This project has involved collaboration with systematists expert in the Heliantheae (Prof. Schilling, Tenessee, USA), the Asteraceae (Assoc Prof. Panero, Texas, USA) and evolutionary biologist Dr Daniel Ortiz-Barrientos (UQ).

In vivo peptide folding

Previous work on preproalbumin and our own work indicate the unusual bifunctional albumin precursor PawS1 undergoes complex processing involving ER-targetting, folding, disulfide bond formation, more trafficking, proteolytic maturation of a cyclic peptide and linear hetero-dimer and eventual storage in protein bodies.

A large body of work by Ikuko Hara-Nishimura and colleagues showed that seed storage proteins are matured by a protease called asparaginyl endopeptidase (AEP). We have demonstrated the same enzyme also matures SFTI-1. We predicted the endoprotease that is required to mature the peptide SFTI-1 out of PawS1 is the same one that perform the ligation reaction that forms it into a peptide ring. Recently we reconstituted this reaction in vitro (Chem. Biol. 2015). During cleavage the perfect conditions would be established for a ligation reaction instead of the usual hydrolysis. AEP seems to be being converged upon by evolution for ligation reactions (Plant Cell 2012). The notion of proteases as ligases in vitro has been reported for over 100 years, but we will attempt to prove that AEP simultaneously cleaves and ligates in vivo.

Our in vivo mutagenesis of PawS1 revealed the importance of some obvious cleavage sites, but others which were potentially involved in its bending preceding the formation of a critical disulfide bond. We are pursuing this with NMR in vitro studies, but also are confirming the mutated residues had no detrimental effect elsewhere upon PawS1 folding.

The goal of this research is to acquire a deeper understanding of peptide processing so we may appreciate the evolutionary significance of the genetic insertion event (e.g. did the insertion need to be in an AEP processed protein), but it will also to assist our NHMRC-funded efforts to manufacture these peptide drugs in the seeds of other plant species.

Hunting for new herbicides

We are exploiting a surprising connection between plants and drugs made for human use as a starting point to discover and develop chemically new herbicides.

The recent Global Herbicide Resistance Challenge conference noted that "no new herbicide mode of action discoveries had been made" and that there are "no new ones coming in the foreseeable future".

This belief was reiterated by Stephen Duke (2012) in a paper entitled 'Why have no new herbicide modes of action appeared in recent years?' saying: 'Herbicides with new modes of action are badly needed to manage the evolution of resistance of weeds to existing herbicides. Yet no major new mode of action has been introduced to the market place for about 20 years'. This problem coupled with decades of over-reliance on glyphosate means there has never been a greater need for new, effective and safe herbicides.

We recently found an exciting connection between plants and drugs made for human use which in turn has led to the discovery of new herbicidal compounds. This project will evaluate some of these to determine their viability as true herbicides plus undertake more fundamental work understanding how they are working in the plant. This project will involve interaction with a team consisting of organic chemists and herbicide physiologists giving exposure to a broad repertoire of skills.

Arabidopsis protein structures

We have just completed an avenue of research that looked at the structure-function relationship between the DNA binding protein VERNALIZATION1 and double stranded DNA.

VERNALIZATION1 is a non-sequence specific DNA binding protein from Arabidopsis that is required for the cold-induced acceleration of flowering time - an epigenetic process called vernalization. Josh Mylne worked on VRN1 extensively at the John Innes Centre in the UK (Science 2002, PNAS 2006). VRN1 was shown to have roles beyond vernalization, localizes throughout the nucleus and remains associated with all five Arabidopsis chromosomes during mitosis.

We have gone on to express the C-terminal region of VRN1 and, with collaborator Prof. Jenny Martin, we have crystallised it (Acta Cryst F 2009) as well as solved its structure at high resolution (image on left, also see PDB file 4i1k). In collaboration with protein NMR spectroscopist Dr Justine Hill, we determined its backbone NMR assignments by triple resonance (Biomol NMR Assign 2012) which combined with the crystal structure allowed us to study DNA binding by VRN1 (JBC 2013).

Defending plants from aphids

Insects eventually find a way to overcome every approach used to protect crops from their ravages. We need to continue to develop new crop protection strategies.

In collaboration with Professor Glenn King (IMB, UQ) and a joint PhD student Md. Shohidul Alam, we are driving the expression of insect-specific peptide toxins in Arabidopsis using the commonly used and strong constitutive promoter 35S, as well as the vascular specific SUCROSE TRANSPORTER 2 (SUC2) promoter (image at left). In this way we aim to confer resistance to insect pests. In particular we will target sap-sucking aphids, which is a class of insects that can circumvent Bt toxin mediated plant protection.

The insect specific peptides in question are derived from those discovered in spiders, whose venom is loaded with peptides that (among other things) has components that can block specific insect neuro-receptors, a property that allows spiders to paralyse prey much larger than they are. Success would mean we have found a chemical-free strategy to protect plants, improving crop yields and securing food production.

A gourd gene expansion event

We are tracing the origin of the genetic event that triggered internal gene expansion and the subsequent creation of ultrastable peptides in members of the gourd family.

The seeds of gourds (Cucurbitaceae) contain a range of disulfide-rich, knotted and ultrastable peptide that has attracted interest from drug designers. One in particular, called TI-2 has been heavily studied; it is a potent trypsin inhibitor (TI), exceptionally stable in plasma assays, capable of penetrating cells, structurally able to tolerate substitutions and additions to its loops and also able to be produced using inteins in E. coli or chemo-enzymatically using trypsin columns. These properties make TI-2 ideal as a scaffold that stabilises peptide drugs as well as an excellent starting point for the design of protease inhibitors.

We recently discovered its biosynthetic route (Plant Cell 2012) so that drugs based on this framework could be made in planta. The proteins we discovered were fascinating. To make TI-2 they appear to have undergone an internal gene expansion event that creates sequences that draw in the function of AEP for its ability to perform peptide ligation reactions. To pursue this case of convergent evolution further, in collaboration with Professor David Craik and a joint PhD student Tunjung Mahatmanto, we worked our way through phylogenetically related specimens (provided by Hanno Schaefer, Harvard University, USA) to find the ancestral unexpanded protein (Mol. Biol. Evol. 2015). In a separate collaboration with Dr Tim Bailey (IMB) we have also started bioinformatic analyses that seek to understand what property of these genes triggered genetic expansion.

Manufacturing drugs in plants

We have performed proof-of-concept work that demonstrates we can genetically modify Arabidopsis with a modified PawS1 gene so that it makes a protease inhibitor lead for of prostate cancer. In collaboration with Professor David Craik, we are assessing this system’s suitability as general machinery for low-cost synthesis of tailor-made drugs in seeds.

Peptides have great potential as drugs, but are currently hampered by the dual-problem of low stability and high cost. Cyclic peptides like SFTI are stable in biological fluids, and although their cyclic backbone has eluded production in simple biological systems, plants offer a solution to their low-cost production. The cost of production is one of the last remaining barriers that needs a tangible solution before peptide-based drugs can enter more widespread use.

Seeds are highly desirable as a target tissue for the production of peptide drugs. Seeds are stable at room temperature, biologically inert, contain almost no water and inside their coat are sterile. There are established systems in place for their production, harvest, storage and transportation. As a biologically self-perpetuating unit, they may be grown using low-cost systems to produce more drug, but can be radiated or roasted so they will not germinate (as poppy seeds are, for example). They do not require the usual infrastructure necessary to manufacture drugs and can benefit nations without the financial strength to import and distribute pharmaceuticals. Finally seeds are already a part of the human diet so could be the ultimate low-cost drug delivery system – the seed becomes the pill.

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