Cloning and characterisation of phospholipase C X-domain containing proteins (PLCXDs)
Members of the phosphoinositide-specific phospholipase C (PI-PLC) enzyme family play a fundamental role in cell signalling pathways by regulating cytosolic calcium and/or the activity of several protein kinases. This thesis reports the identification, molecular cloning and characterisation of a pote...
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2015
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ftstandrewserep:oai:research-repository.st-andrews.ac.uk:10023/7033 2023-07-02T03:32:10+02:00 Cloning and characterisation of phospholipase C X-domain containing proteins (PLCXDs) Gellatly, Steven Alexander Cramb, Gordon 259 2015-07-27T11:53:01Z application/pdf http://hdl.handle.net/10023/7033 en eng University of St Andrews The University of St Andrews http://hdl.handle.net/10023/7033 QP609.P555G4 Phospholipase C Thesis Doctoral PhD Doctor of Philosophy 2015 ftstandrewserep 2023-06-13T18:26:06Z Members of the phosphoinositide-specific phospholipase C (PI-PLC) enzyme family play a fundamental role in cell signalling pathways by regulating cytosolic calcium and/or the activity of several protein kinases. This thesis reports the identification, molecular cloning and characterisation of a potential seventh sub-class of the PI-PLC enzyme family, the phospholipase C X-domain containing proteins (PLCXDs), which contain only an X domain in their structure. Comparative sequence analysis has identified at least three PLCXD isoforms in the human and mouse genomes (PLCXDs 1, 2 and 3), and at least four isoforms in the European eel (PLCXDs 1-4). Key amino acid residues responsible for the catalytic properties of PI-PLCs were found to be conserved in human, mouse and eel PLCXDs 1, 2 and 3, but were absent in the sequence of eel PLCXD4. PLCXD isoforms displayed unique tissue-specific expression profiles and some similarities between species. Interestingly, in mouse PLCXD1-3 mRNA were found to be predominantly expressed in the brain, however this is yet to be confirmed in humans. Analysis of in situ hybridisation data in mice revealed each PLCXD to be localised in neurons within different brain regions, highly suggestive of unique roles in brain function. Furthermore, the levels of PLCXD3 protein were reduced by more than 99% in cerebella samples from a mouse model of neurodegeneration (Harlequin mouse) compared to control mice. Human PLCXD1, 2 and 3 were found to increase phosphoinositide turnover when overexpressed in the HeLa cell line, and recombinant PLCXD3, purified to homogeneity from E. coli, was found to interact with various phosphoinositides including PI(4,5)P₂. ³¹P-NMR analysis of PI(4,5)P₂ and PI before and after the addition of PLCXD3 purified from HeLa cells and E. coli revealed no difference in the ³¹P spectra whereas expected chemical shifts were seen following the addition of purified bacterial PI-PLC. Significant formation of inclusion bodies was noted when human PLCXDs 1, 2 and 3 were expressed as ... Doctoral or Postdoctoral Thesis European eel University of St Andrews: Digital Research Repository |
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Open Polar |
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University of St Andrews: Digital Research Repository |
op_collection_id |
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language |
English |
topic |
QP609.P555G4 Phospholipase C |
spellingShingle |
QP609.P555G4 Phospholipase C Gellatly, Steven Alexander Cloning and characterisation of phospholipase C X-domain containing proteins (PLCXDs) |
topic_facet |
QP609.P555G4 Phospholipase C |
description |
Members of the phosphoinositide-specific phospholipase C (PI-PLC) enzyme family play a fundamental role in cell signalling pathways by regulating cytosolic calcium and/or the activity of several protein kinases. This thesis reports the identification, molecular cloning and characterisation of a potential seventh sub-class of the PI-PLC enzyme family, the phospholipase C X-domain containing proteins (PLCXDs), which contain only an X domain in their structure. Comparative sequence analysis has identified at least three PLCXD isoforms in the human and mouse genomes (PLCXDs 1, 2 and 3), and at least four isoforms in the European eel (PLCXDs 1-4). Key amino acid residues responsible for the catalytic properties of PI-PLCs were found to be conserved in human, mouse and eel PLCXDs 1, 2 and 3, but were absent in the sequence of eel PLCXD4. PLCXD isoforms displayed unique tissue-specific expression profiles and some similarities between species. Interestingly, in mouse PLCXD1-3 mRNA were found to be predominantly expressed in the brain, however this is yet to be confirmed in humans. Analysis of in situ hybridisation data in mice revealed each PLCXD to be localised in neurons within different brain regions, highly suggestive of unique roles in brain function. Furthermore, the levels of PLCXD3 protein were reduced by more than 99% in cerebella samples from a mouse model of neurodegeneration (Harlequin mouse) compared to control mice. Human PLCXD1, 2 and 3 were found to increase phosphoinositide turnover when overexpressed in the HeLa cell line, and recombinant PLCXD3, purified to homogeneity from E. coli, was found to interact with various phosphoinositides including PI(4,5)P₂. ³¹P-NMR analysis of PI(4,5)P₂ and PI before and after the addition of PLCXD3 purified from HeLa cells and E. coli revealed no difference in the ³¹P spectra whereas expected chemical shifts were seen following the addition of purified bacterial PI-PLC. Significant formation of inclusion bodies was noted when human PLCXDs 1, 2 and 3 were expressed as ... |
author2 |
Cramb, Gordon |
format |
Doctoral or Postdoctoral Thesis |
author |
Gellatly, Steven Alexander |
author_facet |
Gellatly, Steven Alexander |
author_sort |
Gellatly, Steven Alexander |
title |
Cloning and characterisation of phospholipase C X-domain containing proteins (PLCXDs) |
title_short |
Cloning and characterisation of phospholipase C X-domain containing proteins (PLCXDs) |
title_full |
Cloning and characterisation of phospholipase C X-domain containing proteins (PLCXDs) |
title_fullStr |
Cloning and characterisation of phospholipase C X-domain containing proteins (PLCXDs) |
title_full_unstemmed |
Cloning and characterisation of phospholipase C X-domain containing proteins (PLCXDs) |
title_sort |
cloning and characterisation of phospholipase c x-domain containing proteins (plcxds) |
publisher |
University of St Andrews |
publishDate |
2015 |
url |
http://hdl.handle.net/10023/7033 |
op_coverage |
259 |
genre |
European eel |
genre_facet |
European eel |
op_relation |
http://hdl.handle.net/10023/7033 |
_version_ |
1770271676583378944 |