Optogenetic activation of heterotrimeric G-proteins by LOV2GIVe, a rationally engineered modular protein
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Abstract
Heterotrimeric G-proteins are signal transducers involved in mediating the action of many natural extracellular stimuli and many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we developed LOV2GIVe, an engineered modular protein that allows the activation of heterotrimeric G-proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, that is metazoan opsins, which are light-activated G-protein-coupled receptors (GPCRs). Instead, LOV2GIVe consists of the fusion of a G-protein activating peptide derived from a non-GPCR regulator of G-proteins to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on July 19 2020, follows.
Summary
Garcia-Marcos et al describe a method to study the activity of heterotrimeric G-proteins. These switches are usually activated via GPCRs and play very important roles in cellular signalling. Investigating their function is often difficult. Therefore the authors have designed an optogenetic tool that activates Gi proteins by blue light based on an engineered LOV2 domain. They demonstrate that activation is specific and that the dark state has a much lower affinity than the light state. The optimization is quite impressive. Overall, this is an interesting and useful tool but some experimental verifications are required.
Essential Revisions
Figure 1 shows binding of the G protein to permanently on or off …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on July 19 2020, follows.
Summary
Garcia-Marcos et al describe a method to study the activity of heterotrimeric G-proteins. These switches are usually activated via GPCRs and play very important roles in cellular signalling. Investigating their function is often difficult. Therefore the authors have designed an optogenetic tool that activates Gi proteins by blue light based on an engineered LOV2 domain. They demonstrate that activation is specific and that the dark state has a much lower affinity than the light state. The optimization is quite impressive. Overall, this is an interesting and useful tool but some experimental verifications are required.
Essential Revisions
Figure 1 shows binding of the G protein to permanently on or off mutant versions of LOV2GIV. Since the G protein is purified, abundant and bound to GST-LOV2GIV, why is it not visible in the ponceau S stained gel?
This figure needs additional controls. Is the interaction with WT LOV2GIV induced by light as shown in the cartoon? Does the interaction lead to increased GTP binding, as shown in the cartoon? Is the binding blocked by GIV residues known to be important for G protein binding as shown in the cartoon structure? Whether or not these controls have been used in the past, they should be done here as well for this particular fusion.
Figure 2A shows binding association (not dissociation as indicated) for the same constructs as in Figure 1. Figure 2B shows GTP hydrolysis but the function of GIV is to stimulate GTP binding, which is just as easy to measure. Again, this figure needs additional controls to show that it is activated by light and relies on key residues.
Figures 3 and 4 shows G protein activation in yeast and HEK293 cells. GIV leads to increased GTP binding but the cell assays do not measure G-alpha-GTP signaling but rather measure release of G-beta-gamma. A direct assay for G-alpha-GTP should be used. The yeast legend and figure do not match and the yeast assays in Figure 3 and 4 use different readouts when both could be used in parallel. A single concentration of a single agonist as a reference is not sufficient when the authors could easily do a concentration-response experiment with an antagonist as a negative control.
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