MCoTI-II Biosynthesis and Applications

Cascales Bolano, Laura (2011). MCoTI-II Biosynthesis and Applications PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Cascales Bolano, Laura
Thesis Title MCoTI-II Biosynthesis and Applications
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2011-05
Thesis type PhD Thesis
Supervisor David Craik
Norelle Daly
Total pages 253
Total colour pages 167
Total black and white pages 86
Subjects 060108 Protein Trafficking
270100 Biochemistry and Cell Biology
030406 Proteins and Peptides
Formatted abstract
    Naturally occurring circular proteins have been found in bacteria, plants, fungi and animals, that, in general, appear to function as defense agents. The cyclotides are the largest family of circular proteins, with more than 200 members characterized so far. Cyclotides contain approximately 30 amino acids, including six conserved cysteine residues that form a cystine knot at the core of their structure. The combination of the cystine knot and the circular backbone defines the cyclic cystine knot (CCK) motif and renders the cyclotides extremely stable. They can be subjected to high temperatures, extremes of pH, or proteolytic enzymes and yet maintain their structure and function. Their structural stability and plasticity to sequence variation makes cyclotides an interesting framework for pharmaceutical applications.

    Cyclotides occur in plants of the Violaceae, Rubiaceae, Fabaceae and Cucurbitaceae families. They are classified into three subfamilies: the Möbius, bracelet or trypsin inhibitor cyclotides. The trypsin inhibitor subfamily comprises only two members, Momordica cochinchinensis trypsin inhibitor I (MCoTI-I), and Momordica cochinchinensis trypsin inhibitor II(MCoTI-II). Despite containing the CCK motif, MCoTI-I/II have significantly different sequences compared to other cyclotides, with only the six cysteine residues conserved, and have much greater sequence similarity to linear squash trypsin inhibitors. MCoTI-II is the most abundant trypsin inhibitor in M. cochinchinensis and is the main focus of this thesis.

    MCoTI-II can penetrate into cells and is not cytotoxic. Consequently, MCoTI-II is a promising candidate in drug design. However, when this thesis project began limited information was available regarding the mechanism by which MCoTI-II penetrates cells, its biosynthesis or its potential to accommodate non-native bioactive sequences within its molecular framework. This information is critical to fully assess the potential of this peptide in drug design and therefore the major aim of this thesis was to perform an interdisciplinary study of MCoTI-II at the genetic, biophysical and cellular levels. Chapter 1 of this thesis sets the scene for these studies by providing a background on the discovery and applications of MCoTI-II and related peptides.

    Chapter 2 focuses on the biosynthesis of MCoTI-II and describes a determination of the partial sequence of the MCoTI-II precursor protein. The clone that was isolated indicated that MCoTI-II is processed in a similar way to other cyclotides, which involves an asparaginyl endopeptidase. This study thus resolved a brewing debate as to whether MCoTI-II should in fact be classified as a cyclotide or not, given its higher sequence similarity to linear knottin peptides than to cyclotides of the Möbius or bracelet subfamilies.

    The aim of Chapter 3 was to explore alternative ways to produce MCoTI-II. An enzymemediated cyclization approach was successfully applied to MCoTI-II, and a range of analogues. However, sequence specific requirements, distant from the ligation site, for cyclization to occur, were identified. These studies provided insight into the interaction of MCoTI-II with trypsin and chymotrypsin.

    Chapter 4 describes an extensive examination of the cellular uptake of MCoTI-II. The affinity of MCoTI-II for model membranes and for cell receptors was examined. Cellular uptake studies were performed in living cells and the entry pathway of MCoTI-II was elucidated. MCoTI-II appears to enter cells by macropinocytosis, but does not interact with model membranes, or with heparan sulfate proteoglycan, a common receptor used by cell penetrating peptides. Interestingly, MCoTI-II did interact with the phospholipid phosphoinositide (4,5)P2, and this interaction might assist in the cellular uptake of MCoTI-II.

    In Chapter 5, the fate of MCoTI-II in the cell was studied and it was shown that MCoTI-II colocalizes with the lysosomes, where the peptide probably undergoes degradation. To avoid degradation of the peptide, a nuclear localization signal was grafted into the MCoTI-II framework to direct the grafted peptide to the nucleus. Preliminary data indicated that the grafted MCoTI-II is delivered to the nucleus in MCF-7 cells.

    In Chapters 6 and 7, MCoTI-II was used to develop novel stable drugs using a peptide grafting approach in which linear epitopes are grafted onto the cyclic peptide framework. In Chapter 6, MCoTI-II was used as a scaffold to graft a sequence that inhibits the interaction of the oncoproteins hdm2, mdm2 and mdmx with p53. In Chapter 7, a β-arrestin 2 domain that activates an inflammatory response was introduced into the MCoTI-II scaffold. Previous studies in our laboratory have shown that this grafted peptide activated macrophage cells and had potential as a vaccine adjuvant. In the current study the mechanism of action of the grafted peptide was determined. The grafting studies did not result in biological activity, but the results presented in this thesis provide valuable information for the design of new grafted MCoTI-II peptides.

    In summary, the studies described in this thesis provide fundamental new knowledge on the biosynthesis and biophysical properties of MCoTI-II, as well as providing valuable information regarding its potential as a template in drug design. MCoTI-II is processed by a similar mechanism to other cyclotides, can be successfully synthesized in vitro, is efficiently taken up by cells, and can accommodate non-native sequences whilst maintaining the native fold. The findings of this thesis are thus of major importance for the future use of MCoTI-II as a stable scaffold in drug design.
Keyword Peptides
Cyclic peptides
Protein engineering
Drug design
Drug delivery
Peptide-membrane interaction
Cell-penetrating peptides

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Created: Mon, 19 Sep 2011, 16:42:36 EST by Ms Laura Cascales Bolano on behalf of Scholarly Communication and Digitisation Service