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Structure of phospholipase Cε reveals an integrated RA1 domain and previously unidentified regulatory elements

Citation

Lyon, Angeline et al. (2021), Structure of phospholipase Cε reveals an integrated RA1 domain and previously unidentified regulatory elements, Dryad, Dataset, https://doi.org/10.5061/dryad.cz8w9gj17

Abstract

Phospholipase Cepsilon (PLCepsilon) generates lipid-derived second messengers in the cardiovascular system at the plasma and perinuclear membranes. It is activated in response to a wide variety of signals, such as those conveyed by Rap1A and Ras, through a mechanism that involves its C-terminal Ras association (RA) domains (RA1 and RA2). However, the complexity and size of PLCepsilon has hindered its structural and functional analysis. In this manuscript, we report the 2.7 Å crystal structure of fragment of PLCepsilon that retains catalytic activity. The strutcure includes the RA1 domain, which forms an integral part of the conserved core. In addition, a highly conserved amphipathic helix in the autoinhibitory X–Y linker is shown to modulate activity in vitro and in cells. The studies provide a structural framework for  the core of this critical cardiovascular enzyme that will allow for a better understanding of its regulation and roles in disease.

Methods

Thermal stability of the PLCepsilon variants was measured using differential scanning fluorimetry. Briefly, a final concentration of 0.5 mg/mL PLCepsilon variant protein was incubated with SYPRO Orange, and the increase in fluorescence was measured as a function of increasing temperature. The data was fit to a Boltzmann sigmoidal function, where the inflection point corresponds to the melting temperature of the protein. 

In vitro PLCepsilon activity was measured using a liposome-based activity assay. In this assay, [3H]-PIP2 is incorporated into PE/PIP2 liposomes, and the amount of free [3H]-IP3 produced is quantified by scintillation counting. Activity was measured at 30 C using a 5 point time course (2-10 min). Control reactions lacked Ca2+ and provide the background [3H]-IP3. Specific activity was calculated as described in 10.1385/1-59259-430-1:67.

Cell-based PLCepsilon activity was measured using the [3H]-inositol phosphates (IPx) accumulation assay. COS-7 cells are transfected with PLCe variants, and metabolically labelled with [3H]-myoinositol. Cells were treated with lithium chloride to inhibit IPx degradation, and [3H]-IPx species isolated by ion exchange chromatography and quantifed by scintillation counting. Protein expression was quantified using by western blot analysis (doi: 10.1126/scisignal.aan1210).

Funding

NIH NHLBI, Award: 1R01HL141076-01

American Heart Association, Award: 18PRE33990057

American Heart Association, Award: 16SDG29920017

American Cancer Society, Award: IRG-14-190-56

National Heart, Lung, and Blood Institute, Award: 1R01HL141076-01

American Heart Association, Award: 18PRE33990057

American Heart Association, Award: 16SDG29920017

American Cancer Society, Award: IRG-14-190-56

NIH NHLBI, Award: 1R01HL141076-01