Subcellular targeting and biosynthesis of cyclotides in plant cells

Conlan, Brendon; Gillon, Amanda D.; Barbeta, Barbara L.; Anderson, Marilyn A.

doi:10.3732/ajb.1100382

Cited by 43 publications

(53 citation statements)

References 49 publications

(67 reference statements)

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“…This is consistent with the discovery that cyclotides are targeted to the vacuole which contains AEPs. That is, the vacuole is the site where processing and cyclization occurs (13). These enzymes are common in plants and have a role in processing seed storage proteins, where they cleave peptide bonds adjacent to Asn residues.…”

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confidence: 99%

“…Cyclotides are expressed as larger precursor proteins containing an endoplasmic reticulum (ER) targeting signal, an N-terminal propeptide, an N-terminal repeat (Ntr) region of 16 -20 amino acids, and a cyclotide domain. The N-terminal regions both target the cyclotide precursor to the vacuole where the protein is post-translationally processed and cyclized (13). The Ntr and cyclotide domains can be repeated up to three times in some cyclotide precursors and the repeated domains contain a short hydrophobic C-terminal region (Ctr) of 3 to 7 amino acids (Fig.…”

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confidence: 99%

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Insights into Processing and Cyclization Events Associated with Biosynthesis of the Cyclic Peptide Kalata B1

Conlan

Colgrave

Gillon

et al. 2012

Journal of Biological Chemistry

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Background: Production of insecticidal and nematocidal cyclic peptides is inefficient in transgenic plants. Results: Efficient cyclization requires cleavage of the N-terminal propeptide from the mature cyclotide domain and a C terminus containing a binding motif. Conclusion:The cyclization motif has Asn at position P1, a small amino acid at position P1Ј, and a Leu at position P2Ј. Significance: Understanding substrate requirements will help produce cyclotides in transgenic plants.

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confidence: 99%

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confidence: 99%

Insights into Processing and Cyclization Events Associated with Biosynthesis of the Cyclic Peptide Kalata B1

Conlan

Colgrave

Gillon

et al. 2012

Journal of Biological Chemistry

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“…The cystine knot domain of kalata B1 is flanked by both an N-and a C-terminal segment. The N-terminal segment, ntr, has been found to target the protein to the vacuole and to be intrinsically unstructured (Conlan et al, 2011;Daly et al, 2016).…”

Section: Cystine Knot Peptidesmentioning

confidence: 99%

Cystine knot growth factors and their functionally versatile proregions

Schwarz

2017

Biological Chemistry

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Abstract:The cystine knot disulfide pattern has been found to be widespread in nature, since it has been detected in proteins from plants, marine snails, spiders and mammals. Cystine knot proteins are secreted proteins. Their functions range from defense mechanisms as toxins, e.g. ion channel or enzyme inhibitors, to hormones, blood factors and growth factors. Cystine knot proteins can be divided into two superordinate groups. (i) The cystine knot peptides, also referred to -with other non-cystine knot proteins -as knottins, with linear and cyclic polypeptide chains. (ii) The cystine knot growth factor family, which is in the focus of this article. The disulfide ring structure of the cystine knot peptides is made up by the half-cystines 1-4 and 2-5, and the threading disulfide bond is formed by the half-cystines, 3-6. In the growth factor group, the disulfides of half-cystines 1 and 4 pass the ring structure formed by the half-cystines 2-5 and 3-6. In this review, special emphasis will be devoted to the growth factor cystine knot proteins and their proregions. The latter have shifted into the focus of scientific interest as their important biological roles are just to be unravelled.

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“…Nine constructs were made to identify the regions from the Oak1 precursor in O. affinis that are responsible for the subcellular targeting of kalata B1. Through this approach, they were able to demonstrate that GFP-tagged cyclotide precursors accumulate in living plant cell vacuoles (Conlan et al, 2011). This suggests that excision and cyclization processes take place in the vacuole where cyclotides are later stored.…”

Section: Strategies For Selective Classes Of Compoundsmentioning

confidence: 98%

“…Recently, subcellular targeting of cyclotides in plant cells was reported by Anderson and colleagues (Conlan et al, 2011). In this work, a combination of confocal microscopy and MALDI-TOF MS techniques were used to examine the targeting of green fluorescent protein (GFP)-tagged constructs expressed transiently in N. benthamiana.…”

Section: Strategies For Selective Classes Of Compoundsmentioning

confidence: 99%

Structure-activity relationships and development of disulfide-rich cyclic peptides as pharmaceutical templates

D’Souza¹

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Peptides are attracting increasing attention from pharmaceutical companies as promising drug leads. Peptides have high specificity and high-affinity potency towards molecular targets as well as low toxicity, making them viable alternatives to small molecules. However, their lack of membrane permeability, bioavailability and in vivo stability hinders most peptides from becoming actual drugs. Although peptides are generally thought to be poor drugs, disulfide-rich cyclic peptides show great potential as pharmaceutical scaffolds in peptide-based drug design because of their exceptional stability.Naturally occurring disulfide-rich cyclic peptides have been found in plants and animals.Cyclotides are among the largest naturally occurring disulfide-rich cyclic peptides discovered so far and have been identified in species of Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae and Solanaceae plant families. Cyclotides are distinguished by the cyclic cystine knot (CCK) motif, which comprises a macrocyclic backbone and three conserved disulfide bonds. The CCK motif provides cyclotides with exceptional stability, making them promising scaffolds for peptide drugs.Cyclotides were initially classified into two main subfamilies, Mӧbius (e.g, kalata B1 and kalata B2) and bracelet (e.g, cycloviolacin O2), based on the presence/absence of a cis-Pro peptide bond.A third subfamily, the trypsin inhibitor subfamily, was later introduced following the discovery of two peptides (MCoTI-I and MCoTI-II) obtained from Momordica cochinchinensis seeds. Other trypsin inhibitors in this family have now been identified. This subfamily is homologous to acyclic squash inhibitors and share little sequence similarity to cyclotides from the other two subfamilies.Recently, cyclotides kalata B1 and MCoTI-II have been shown to internalise into cells and were therefore named cyclic cell-penetrating peptides (CCPPs). Another CCPP is SFTI-1, a sunflower trypsin inhibitor that comprises a single disulfide-bond and has been used as a scaffold in drug design.The aim of my Ph.D. thesis was to understand the mechanism of action and unravel the cellpenetrating properties of disulfide-rich cyclic peptides. Chapter 1 provides an introduction on disulfide-rich cyclic peptides with an emphasis on their structural features and their biological activities, including membrane-binding and cell-penetrating properties, as well as their potential as drug design scaffolds in various therapeutic applications.Chapter 2 describes the materials and methods used to carry out the experiments in this thesis, which include methods to synthesise peptides that were studied, and biological assays used to evaluate them. In addition biophysical methodologies to evaluate structural features, peptide iv internalisation and peptide-membrane interactions are also detailed.Chapter 3 is a research article published in the European Journal of Medicinal Chemistry (2014) that focuses on the structural characteristics affecting the cell-penetrating properties of disulfide-rich cyclic peptides,...

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Subcellular targeting and biosynthesis of cyclotides in plant cells

Abstract: The vacuolar location of cyclotides supports our hypothesis that the vacuolar processing enzyme, asparaginyl endoproteinase, has a pivotal role in excision and cyclization from cyclotide precursors.

Cited by 43 publications

References 49 publications

Insights into Processing and Cyclization Events Associated with Biosynthesis of the Cyclic Peptide Kalata B1

Insights into Processing and Cyclization Events Associated with Biosynthesis of the Cyclic Peptide Kalata B1

Cystine knot growth factors and their functionally versatile proregions

Structure-activity relationships and development of disulfide-rich cyclic peptides as pharmaceutical templates

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