Interactions of phosphoinositide specific phospholipase C with a lipid layer for structural and functional studies
Author(s)
Arduin, Alessia
Type
Thesis
Abstract
Phosphoinositide-specific phospholipase C (PLC) is an intensively studied family
of enzymes constituting a junction between trans-membrane signal transduction
processes and phosphoinositide lipid signalling. PLCs are activated in response
to stimulation of cell surface receptors at the plasma membrane, and the
signals are carried downstream by other transducers. PLCs catalyse the hydrolysis
of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to diacylglycerol and
inositol 1,4,5-trisphosphate, which are both well known intracellular second messengers.
This study mainly focused on the PLCε sub-family which is closely linked
to the Ras oncogene and may play a role in tumorigenesis and development. The
functions and regulatory mechanism of PLCε are not yet understood in detail. To
address these issues activity and structural studies were performed.
Activity studies were carried out in vivo using cell lines and in vitro using
lipid vesicles in a model system. The latter was designed to study protein-protein
and lipid-protein interactions using PLC purified to homogeneity and guanosine
triphosphatases (GTPases) prenylated in vitro. Evidence was found for a direct
interaction between the GTPases and the PLC that mediated activation of the phospholipase.
Furthermore, the correlation between PLC activity and substrate presentation
in lipid vesicles of various sizes and lipid compositions was analysed. For the
first time, PLC activity was found to depend upon the electrostatic potential and the
stored elastic curvature stress of the lipid bilayer of the vesicles.
The binding and the activation process between GTPase (using H-Ras) and
PLCε was also investigated at a molecular level in vitro. Functional studies were carried out using Förster resonance energy transfer (FRET) to determine if PLCε
undergoes a conformational change upon H-Ras binding. This would distinguish
whether conformational change or translocation of PLCε to the membrane interface
(where GTPases are localised) is most likely to be the key event during PLCε
activation; no conformational change was observed.
Electron crystallographic structural studies, in which two-dimensional protein
crystals are grown on a lipid monolayer followed by electron microscopy, were attempted.
The aim was to retrieve structural information in a functional state that
resembles the natural one. Protein and lipid monolayer compositions (lipid proportions,
lipid concentration, protein concentration and incubation time) were screened
to identify conditions where specific protein-lipid interaction would favour twodimensional
crystal formation. The protein was expressed with a His6-tag that allowed
specific binding to nickel chelating lipids included in the lipid monolayer.
In addition, catalytically inactive PLCε mutants were generated and their ability
to bind PtdIns(4,5)P2, and thereby to drive the crystallisation process, was investigated.
Conditions that led to protein-lipid binding, but not to two-dimensional
crystallisation, were identified.
of enzymes constituting a junction between trans-membrane signal transduction
processes and phosphoinositide lipid signalling. PLCs are activated in response
to stimulation of cell surface receptors at the plasma membrane, and the
signals are carried downstream by other transducers. PLCs catalyse the hydrolysis
of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to diacylglycerol and
inositol 1,4,5-trisphosphate, which are both well known intracellular second messengers.
This study mainly focused on the PLCε sub-family which is closely linked
to the Ras oncogene and may play a role in tumorigenesis and development. The
functions and regulatory mechanism of PLCε are not yet understood in detail. To
address these issues activity and structural studies were performed.
Activity studies were carried out in vivo using cell lines and in vitro using
lipid vesicles in a model system. The latter was designed to study protein-protein
and lipid-protein interactions using PLC purified to homogeneity and guanosine
triphosphatases (GTPases) prenylated in vitro. Evidence was found for a direct
interaction between the GTPases and the PLC that mediated activation of the phospholipase.
Furthermore, the correlation between PLC activity and substrate presentation
in lipid vesicles of various sizes and lipid compositions was analysed. For the
first time, PLC activity was found to depend upon the electrostatic potential and the
stored elastic curvature stress of the lipid bilayer of the vesicles.
The binding and the activation process between GTPase (using H-Ras) and
PLCε was also investigated at a molecular level in vitro. Functional studies were carried out using Förster resonance energy transfer (FRET) to determine if PLCε
undergoes a conformational change upon H-Ras binding. This would distinguish
whether conformational change or translocation of PLCε to the membrane interface
(where GTPases are localised) is most likely to be the key event during PLCε
activation; no conformational change was observed.
Electron crystallographic structural studies, in which two-dimensional protein
crystals are grown on a lipid monolayer followed by electron microscopy, were attempted.
The aim was to retrieve structural information in a functional state that
resembles the natural one. Protein and lipid monolayer compositions (lipid proportions,
lipid concentration, protein concentration and incubation time) were screened
to identify conditions where specific protein-lipid interaction would favour twodimensional
crystal formation. The protein was expressed with a His6-tag that allowed
specific binding to nickel chelating lipids included in the lipid monolayer.
In addition, catalytically inactive PLCε mutants were generated and their ability
to bind PtdIns(4,5)P2, and thereby to drive the crystallisation process, was investigated.
Conditions that led to protein-lipid binding, but not to two-dimensional
crystallisation, were identified.
Date Issued
2011-09
Date Awarded
2011-10
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Katan, Matilda
Morris, Edward
Ces, Oscar
Creator
Arduin, Alessia
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)