Browsing by Author "Hendrikse, Megan"

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    Interactions of GPR54 and GPR147 receptors with RF-amide ligands
    (2014) Hendrikse, Megan; Katz, Arieh; Millar, Robert
    G protein-coupled receptors play a key role in cellular signaling by transducing extracellular signals via G proteins to elicit intracellular responses. Studies have provided evidence supporting the role of the GPCR GPR54 and its cognate peptide ligand, kisspeptin (an RFamide peptide), in the regulation of reproduction. Kisspeptin and GPR54 play a critical role in the control of the hypothalamic-pituitary-gonadal axis by regulating gonadotropin-releasing hormone secretion. Despite the physiological importance of GPR54/kisspeptin signalling, the GRP54 residues important for receptor activation and signalling have not been extensively investigated. Another hypothalamic peptide, gonadotropin inhibiting hormone (also known as RFamide-related peptide), which interacts with the GPCR GPR147, has been found to inhibit GnRH-induced gonadotropin release and is therefore also of importance in control of the HPG axis. As many of the RFamide and RFamide-related receptors and ligands can be promiscuous, there is the potential for crosstalk between the GPR54/kisspeptin and GRP147/RFRP systems (or other RFamides) which may be of importance in the regulation of reproduction. GPR54 chimeras and point mutants were constructed in order to investigate the residues important for kisspeptin binding and receptor activation. The data obtained indicate that the acidic residues within the extracellular loops of GPR54 contribute to cell surface receptor expression and play a role in receptor signalling. In order to investigate the interactions of kisspeptin/RFRP peptides at GPR147 and GPR54, binding and activation of these receptors was studied with a range of ligands and their analogs. In addition to RFRP and its analogs, kisspeptin and several kisspeptin analogs were found to act as agonists at GRP147. In contrast, of all the ligands tested, only kisspeptin was able to bind to GPR54 with high affinity and elicit a response, thus indicating that GPR54 has high specificity for kisspeptin in contrast to the more promiscuous GPR147. These data demonstrate the therapeutic potential of kisspeptin analogs, for the inhibition of gonadotropin secretion and treatment of sex steroid hormone disease. In addition, these data have identified ligand and receptor residues important for binding and activation of GRP54/GRP147 which may aid development of new analogs targeting these receptors and highlighted the importance of testing these analogs for receptor specificity.
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    Optimization and characterisation of plant produced Human Papillomavirus pseudovirions in Nicotiana benthamiana
    (2020) Adams, Ayesha; Hitzeroth, Inga; Hendrikse, Megan; Rybicki, Ed
    Human papillomavirus (HPV) is known to be the cause of anogenital and oropharyngeal cancers as well as genital and common warts. There are currently three prophylactic virus-like particle (VLP) based vaccines. These vaccines, however, do not offer protection against all HPV strains and cannot act therapeutically and so further vaccine development is still needed. The burden of HPV is also highest in low-income countries for which the vaccine costs are still quite high, and therefore alternative methods of vaccine production and testing are needed. HPV pseudovirions (PsVs) are synthetic viral particles that are made up of the L1 major and L2 minor HPV capsid proteins and encapsidate up to 8Kb of pseudogenome DNA without the need of an encapsidation signal. HPV PsVs are used to test neutralising antibodies elicited by vaccines, for studying the virus life cycle, and potentially for delivery of therapeutic DNA vaccines. HPV PsVs are typically produced in mammalian cells; however, it has recently been shown that HPV PsVs can be produced in plants, a potentially safer, cheaper and more easily scalable means of production. While, a current problem with plant HPV PsV production is low yields, research has shown that using pseudogenome DNAs between 5-7Kb increases yields of papillomavirus PsVs in mammalian cells. Therefore, the objective of this study was to determine the optimal pseudogenome size for encapsidation by plant produced PsVs, in order to increase the amount of PsVs in a sample as opposed to VLPs. Pseudogenome constructs encoding Enhanced Green Fluorescent Protein (EGFP )and ranging in size from 4.8Kb – 7.8kb were cloned into a geminivirus-derived replicating vector, transformed into Agrobacterium tumefaciens and then infiltrated into Nicotiana benthamiana along with plant expression vectors encoding the HPV 35 L1 and L2 capsid proteins. Particles were purified by iodixanol density gradient ultracentrifugation and the 27% and 33% fractions of this gradient analysed. Transmission electron microscopy (TEM) was used to confirm particle assembly and L1 expression was quantified by ELISA. Particles were disrupted with proteinase K and quantitative PCR was used to quantify the encapsidated DNA. Ratios of encapsidated DNA to L1 capsid protein were calculated for each of the PsV samples with different sized pseudogenomes, to account for batch-to-batch variation and as an approximation of which size pseudogenome is better encapsidated. Infective ability of the particles was analysed by incubating the PsVs onto HEK293TT cells and then checking for DNA delivery and protein expression by measuring EGFP expression by Western blots. The results showed that PsVs are found predominantly in the 27% fraction of the iodixanol gradient whereas the 33% fraction of the gradient appears to only contain VLPs. The data also indicated that the smaller pseudogenomes, were packaged more efficiently into PsVs as higher concentrations of encapsidated DNA and higher levels of EGFP expression were obtained when the 4.8Kb pseudogenome was used, compared to when the larger 5.8 - 7.8Kb pseudogenomes were used. Thus, the results showed that smaller pseudogenomes, around 4.8Kb, should be used for the plant production of HPV 35 PsVs as they are better packaged than larger pseudogenomes and thereby produce higher yields of functional PsVs.