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Regulation of PLCβ2 by the electrostatic and mechanical properties of lipid bilayers.

Arduin A, Gaffney PR, Ces O - Sci Rep (2015)

Bottom Line: However, the regulatory mechanism of PLC is not yet understood in detail.Evidence was found for a direct interaction between PLC and the GTPases that mediate phospholipase activation.PLC activity was found to depend upon the electrostatic potential and the stored curvature elastic stress of the lipid membranes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Biology, Department of Chemical Biology, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom.

ABSTRACT
Phosphoinositide-specific phospholipase C (PLC) is an important family of enzymes constituting a junction between phosphoinositide lipid signaling and the trans-membrane signal transduction processes that are crucial to many living cells. However, the regulatory mechanism of PLC is not yet understood in detail. To address this issue, activity studies were carried out using lipid vesicles in a model system that was specifically designed to study protein-protein and lipid-protein interactions in concert. Evidence was found for a direct interaction between PLC and the GTPases that mediate phospholipase activation. Furthermore, for the first time, the relationships between PLC activity and substrate presentation in lipid vesicles of various sizes, as well as lipid composition and membrane mechanical properties, were analyzed. PLC activity was found to depend upon the electrostatic potential and the stored curvature elastic stress of the lipid membranes.

No MeSH data available.


Related in: MedlinePlus

Activation of PLCβ2 by GTP bound prenylated Rac2 in the cell-free system.The bar chart reports the average activity measured in duplicate samples (± the s.e.m.) upon sample incubation at 30 °C for 45 min. The samples contained: purified PLCβ2 (2–803 AA) (0.5 ng/μl, equivalent to 5.4 nM), DOPE: PtdIns (4,5)P2/[3H]-PtdIns (4,5)P2 lipid vesicles 16:1 mol/mol, 300 nM free calcium, 100 μM GDP (blue bars), 100 μM GTPγS (red bars), and 1.6 ng/μl GG-Rac2.
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f2: Activation of PLCβ2 by GTP bound prenylated Rac2 in the cell-free system.The bar chart reports the average activity measured in duplicate samples (± the s.e.m.) upon sample incubation at 30 °C for 45 min. The samples contained: purified PLCβ2 (2–803 AA) (0.5 ng/μl, equivalent to 5.4 nM), DOPE: PtdIns (4,5)P2/[3H]-PtdIns (4,5)P2 lipid vesicles 16:1 mol/mol, 300 nM free calcium, 100 μM GDP (blue bars), 100 μM GTPγS (red bars), and 1.6 ng/μl GG-Rac2.

Mentions: Control experiments showed that the basal activity of PLCβ2 (2–803 AA) in the absence of GTPase was unaffected by the presence of either GDP or GTPγS (columns 1 and 2, Fig. 2), and that the addition of un-prenylated Rac2 to the cell-free system caused a negligible increase in PLC activity. This did not change in the presence of GDP (Fig. 2, columns 1 and 3) or of GTPγS (Fig. 2, columns 2 and 4). When geranylgeranylated Rac2 (GG-Rac2) was added, the PLC activity measured in the presence of GDP was again comparable to the activity measured in the presence of GDP plus un-prenylated Rac2. However, in the presence of GTPγS, PLC activity was 3-fold higher than that measured in the presence of GDP and GG-Rac2 (Fig. 2, columns 5 and 6). The response curve shows that the range of concentrations of PLCβ2 (2–803 AA) tested were in the linear range (Fig. 3).


Regulation of PLCβ2 by the electrostatic and mechanical properties of lipid bilayers.

Arduin A, Gaffney PR, Ces O - Sci Rep (2015)

Activation of PLCβ2 by GTP bound prenylated Rac2 in the cell-free system.The bar chart reports the average activity measured in duplicate samples (± the s.e.m.) upon sample incubation at 30 °C for 45 min. The samples contained: purified PLCβ2 (2–803 AA) (0.5 ng/μl, equivalent to 5.4 nM), DOPE: PtdIns (4,5)P2/[3H]-PtdIns (4,5)P2 lipid vesicles 16:1 mol/mol, 300 nM free calcium, 100 μM GDP (blue bars), 100 μM GTPγS (red bars), and 1.6 ng/μl GG-Rac2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525138&req=5

f2: Activation of PLCβ2 by GTP bound prenylated Rac2 in the cell-free system.The bar chart reports the average activity measured in duplicate samples (± the s.e.m.) upon sample incubation at 30 °C for 45 min. The samples contained: purified PLCβ2 (2–803 AA) (0.5 ng/μl, equivalent to 5.4 nM), DOPE: PtdIns (4,5)P2/[3H]-PtdIns (4,5)P2 lipid vesicles 16:1 mol/mol, 300 nM free calcium, 100 μM GDP (blue bars), 100 μM GTPγS (red bars), and 1.6 ng/μl GG-Rac2.
Mentions: Control experiments showed that the basal activity of PLCβ2 (2–803 AA) in the absence of GTPase was unaffected by the presence of either GDP or GTPγS (columns 1 and 2, Fig. 2), and that the addition of un-prenylated Rac2 to the cell-free system caused a negligible increase in PLC activity. This did not change in the presence of GDP (Fig. 2, columns 1 and 3) or of GTPγS (Fig. 2, columns 2 and 4). When geranylgeranylated Rac2 (GG-Rac2) was added, the PLC activity measured in the presence of GDP was again comparable to the activity measured in the presence of GDP plus un-prenylated Rac2. However, in the presence of GTPγS, PLC activity was 3-fold higher than that measured in the presence of GDP and GG-Rac2 (Fig. 2, columns 5 and 6). The response curve shows that the range of concentrations of PLCβ2 (2–803 AA) tested were in the linear range (Fig. 3).

Bottom Line: However, the regulatory mechanism of PLC is not yet understood in detail.Evidence was found for a direct interaction between PLC and the GTPases that mediate phospholipase activation.PLC activity was found to depend upon the electrostatic potential and the stored curvature elastic stress of the lipid membranes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Biology, Department of Chemical Biology, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom.

ABSTRACT
Phosphoinositide-specific phospholipase C (PLC) is an important family of enzymes constituting a junction between phosphoinositide lipid signaling and the trans-membrane signal transduction processes that are crucial to many living cells. However, the regulatory mechanism of PLC is not yet understood in detail. To address this issue, activity studies were carried out using lipid vesicles in a model system that was specifically designed to study protein-protein and lipid-protein interactions in concert. Evidence was found for a direct interaction between PLC and the GTPases that mediate phospholipase activation. Furthermore, for the first time, the relationships between PLC activity and substrate presentation in lipid vesicles of various sizes, as well as lipid composition and membrane mechanical properties, were analyzed. PLC activity was found to depend upon the electrostatic potential and the stored curvature elastic stress of the lipid membranes.

No MeSH data available.


Related in: MedlinePlus