Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis.

The cyclic miniprotein Momordica cochinchinensis Trypsin Inhibitor II (MCoTI-II) (34 amino acids) is a potent trypsin inhibitor (TI) and a favored scaffold for drug design. We have cloned the corresponding genes and determined that each precursor protein contains a tandem series of cyclic TIs terminating with the more commonly known, and potentially ancestral, acyclic TI. Expression of the precursor protein in Arabidopsis thaliana showed that production of the cyclic TIs, but not the terminal acyclic TI, depends on asparaginyl endopeptidase (AEP) for maturation. The nature of their repetitive sequences and the almost identical structures of emerging TIs suggest these cyclic peptides evolved by internal gene amplification associated with recruitment of AEP for processing between domain repeats. This is the third example of similar AEP-mediated processing of a class of cyclic peptides from unrelated precursor proteins in phylogenetically distant plant families. This suggests that production of cyclic peptides in angiosperms has evolved in parallel using AEP as a constraining evolutionary channel. We believe this is evolutionary evidence that, in addition to its known roles in proteolysis, AEP is especially suited to performing protein cyclization.

Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins.

Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule.