Year: 2015
Type: dissertation
Abstract: Members of the phosphoinositide-specific phospholipase C (PI-PLC) enzyme family \nplay a fundamental role in cell signalling pathways by regulating cytosolic calcium \nand/or the activity of several protein kinases. This thesis reports the identification, \nmolecular cloning and characterisation of a potential seventh sub-class of the PI-PLC \nenzyme family, the phospholipase C X-domain containing proteins (PLCXDs), which \ncontain only an X domain in their structure. Comparative sequence analysis has \nidentified at least three PLCXD isoforms in the human and mouse genomes (PLCXDs 1, \n2 and 3), and at least four isoforms in the European eel (PLCXDs 1-4). Key amino acid \nresidues responsible for the catalytic properties of PI-PLCs were found to be conserved \nin human, mouse and eel PLCXDs 1, 2 and 3, but were absent in the sequence of eel \nPLCXD4. PLCXD isoforms displayed unique tissue-specific expression profiles and some \nsimilarities between species. Interestingly, in mouse PLCXD1-3 mRNA were found to \nbe predominantly expressed in the brain, however this is yet to be confirmed in \nhumans. Analysis of in situ hybridisation data in mice revealed each PLCXD to be \nlocalised in neurons within different brain regions, highly suggestive of unique roles in \nbrain function. Furthermore, the levels of PLCXD3 protein were reduced by more than \n99% in cerebella samples from a mouse model of neurodegeneration (Harlequin \nmouse) compared to control mice. Human PLCXD1, 2 and 3 were found to increase \nphosphoinositide turnover when overexpressed in the HeLa cell line, and recombinant \nPLCXD3, purified to homogeneity from E. coli, was found to interact with various \nphosphoinositides including PI(4,5)P₂. ³¹P-NMR analysis of PI(4,5)P₂ and PI before and \nafter the addition of PLCXD3 purified from HeLa cells and E. coli revealed no difference \nin the ³¹P spectra whereas expected chemical shifts were seen following the addition \nof purified bacterial PI-PLC. Significant formation of inclusion bodies was noted when \nhuman PLCXDs 1, 2 and 3 were expressed as recombinant proteins in E. coli. Different \nstrategies aimed at optimising the expression of recombinant PLCXD1, 2 and 3, \nincluding the use of different fusion proteins and screening expression in E. coli, \nmammalian and insect cells had limited success, with the best soluble expression only \nseen with PLCXD3 in insect cells. Attempts to scale-up the purification of PLCXD3 from \ninsect cells to provide sufficient protein for enzyme assays and crystal screens were \nunsuccessful. The results presented herein suggest that these novel proteins possess \ndistinct and as yet uncharacterised tissue-specific roles in cell physiology.
Author: Steven A. Gellatly
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1438472422