The Premiers prize for best " protein-focused abstract " at the recent ASMR Queensland Postgraduate Student Conference was won by Rodrigo Morales of the Alewood group. The prize was sponsored by the Queensland Protein Group (QPG)
Copy of Abstract below -
Role of the N-terminal AVITG for the structure and pharmacological function of the Prokineticin Bv8 in the SH-SY5Y neuroblastoma cell line
Rodrigo Morales, Thomas Durek, Irina Vetter, Mehdi Mobli, Glenn King, Richard Lewis and Paul Alewood
Institute for Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane, QLD 4072
Bv8 is a cysteine-rich protein originally isolated from skin secretions of the European frog, Bombina variegata. Bv8 and other members of the Prokineticin family (PK) are potent inducers of constitutive neuronal and inflammatory cell migration, olfactory morphogenesis, nociception, circadian rhythms and angiogenesis. In disease, Prokineticins and their receptors have been implicated in the development of aggressive forms of neuroblastoma1, myeloid-dependant tumors2 and hepato-carcinomas3. Recently, the functionalities of Bv8 were linked to its conserved AVITG N-terminal domain but detailed structural studies are currently hampered by extremely low yields after isolation from biological sources. Here we used a total chemical synthesis approach to obtain multi-milligram amounts of Bv8 and designed analogues. The role of the conserved N-terminal AVITG domain was further explored in a novel high-throughput functional Ca2+ influx assays using the PK responsive SH-SY5Y neuroblastoma cell line and in NMR experiments using Bv8 and the N-terminal truncated analogue desAVITG-Bv8.
The full length reduced Bv8 of 77 amino acids was smoothly assembled from three unprotected peptide segments using native chemical ligation (NCL) to covalently join the segments in solution. The peptides were prepared by Boc solid phase peptide synthesis (SPPS) chemistry, cleaved by anhydrous HF and purified by RP-HPLC. The individual segments (Bv8 (1-18), (19-58) and (59-77) were linked together by two consecutive NCL reactions yielding the full-length 77-aa. Analogs of Bv8 with modifications in the N-terminal AVITG region were prepared in a similar manner. Folding and disulfide bond formation was achieved cleanly by diluting the fully reduced peptide into folding buffer (50mM of Tris-HCl buffer, 500mM GnHCl, 1mM GSH, 0.2mM GSSG) for 72 hours. Formation of the 5 disulfide bonds and concomitant loss of 10 protons was followed by LC-MS. The purified full-length Bv8 and the desAVITG-Bv8 proteins were analysed by 2D H1 NMR spectroscopy. The function of Bv8 was measured in SH-SY5Y human neuroblastoma cells pre-incubated with a Ca2+ sensitive dye. Real-time readings were taken every second for 300 seconds after addition of Bv8 or analogues using the FLIPRTETRA fluorometric imaging system. The data was analysed using the automated analysis software ScreenWorks 2.0.
Synthetic Bv8 and desAVITG-Bv8 were obtained in high yield and purity. The established synthetic strategy should permit preparation of various Bv8 analogs including proteins carrying non-proteinogenic functionalities. Bv8 increased the intracellular Ca2+ influx in a concentration-dependant manner (EC50 273pM) comparable to the native peptide. However, desAVITG-Bv8 with a similar folding profile and three-dimensional structure was unable to elicit either agonist or antagonist activity. Thus it appears that the role of the AVITG domain does not affect the structure of Bv8 but either stabilizes receptor binding of the toxin framework or provides a direct interaction with the Prokineticin receptor. We believe the use of total chemical synthesis and the establishment of a screening platform for the function of Bv8 will give us the exciting opportunity to further explore the functional role of the AVITG domain, and ultimately develop peptide antagonists as anticancer therapies or molecular tools for the study of Prokineticins in many key cellular processes.
References:
1 E. S. Ngan, F. Y. Sit, K. Lee et al., Clin Cancer Res 13 (3), 868 (2007).
2 F. Shojaei, X. Wu, C. Zhong et al., Nature 450 (7171), 825 (2007).
3 Q. Li, B. Xu, L. Fu et al., J Exp Clin Cancer Res 25 (3), 403 (2006).