Dual-controlled optogenetic system for the rapid down-regulation of protein levels in mammalian cells

Abstract Optogenetic switches are emerging molecular tools for studying cellular processes as they offer higher spatiotemporal and quantitative precision than classical, chemical-based switches. Light-controllable gene expression systems designed to upregulate protein expression levels meanwhile show performances superior to their chemical-based counterparts. However, systems to reduce protein levels with similar efficiency are lagging behind. Here, we present a novel two-component, blue light-responsive optogenetic OFF switch (‘Blue-OFF’), which enables a rapid and quantitative down-regulation of a protein upon illumination. Blue-OFF combines the first light responsive repressor KRAB-EL222 with the protein degradation module B-LID (blue light-inducible degradation domain) to simultaneously control gene expression and protein stability with a single wavelength. Blue-OFF thus outperforms current optogenetic systems for controlling protein levels. The system is described by a mathematical model which aids in the choice of experimental conditions such as light intensity and illumination regime to obtain the desired outcome. This approach represents an advancement of dual-controlled optogenetic systems in which multiple photosensory modules operate synergistically. As exemplified here for the control of apoptosis in mammalian cell culture, the approach opens up novel perspectives in fundamental research and applications such as tissue engineering

Factors That Control the Chemistry of the LOV Domain Photocycle

Algae, plants, bacteria and fungi contain Light-Oxygen-Voltage (LOV) domains that function as blue light sensors to control cellular responses to light. All LOV domains contain a bound flavin chromophore that is reduced upon photon absorption and forms a reversible, metastable covalent bond with a nearby cysteine residue. In Avena sativa LOV2 (AsLOV2), the photocycle is accompanied by an allosteric conformational change that activates the attached phototropin kinase in the full-length protein. Both the conformational change and formation of the cysteinyl-flavin adduct are stabilized by the reduction of the N5 atom in the flavin’s isoalloxazine ring. In this study, we perform a mutational analysis to investigate the requirements for LOV2 to photocycle. We mutated all the residues that interact with the chromophore isoalloxazine ring to inert functional groups but none could fully inhibit the photocycle except those to the active-site cysteine. However, electronegative side chains in the vicinity of the chromophore accelerate the N5 deprotonation and the return to the dark state. Mutations to the N414 and Q513 residues identify a potential water gate and H2O coordination sites. These residues affect the electronic nature of the chromophore and photocycle time by helping catalyze the N5 reduction leading to the completion of the photocycle. In addition, we demonstrate that dehydration leads to drastically slower photocycle times. Finally, to investigate the requirements of an active-site cysteine for photocycling, we moved the nearby cysteine to alternative locations and found that some variants can still photocycle. We propose a new model of the LOV domain photocycle that involves all of these components.</p

Chromophore and Photocycle parameters of <i>As</i>LOV2 variants.

†<p>Samples were air dried for 48 hours.</p

UV-Vis absorbance changes upon photoexcitation and recovery for selected <i>As</i>LOV2 variants.

<p>Single exponential fits to the time dependent data are shown in red.</p

Effects on <i>As</i>LOV2 side chains during the photocycle.

<p>(A) Side chain positions in the chromophore binding pocket in the dark and light state crystal structures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087074#pone.0087074-Halavaty1" target="_blank">[32]</a> (B) Chemical processes that occur to the FMN chromophore during the photocycle.</p

<i>As</i>LOV2 photocycle chemistry.

<p>Photon absorption results in the FMN being excited into a singlet then triplet state. The N5 of FMN becomes a strong nucleophile and removes the proton from the nearby C450 to form a covalent bond with the FMN C4a atom. Reduction of the FMNH state is inhibited by the inability of water to readily enter the chromophore binding pocket and catalyze the transfer of the proton back to the cysteine until a conformational change occurs involving residues N414 and Q513. The covalent bond is broken and the protein returns to the dark state.</p

Helical Contributions Mediate Light-Activated Conformational Change in the LOV2 Domain of Avena sativa Phototropin 1

Algae, plants, bacteria, and fungi contain flavin-binding light-oxygen-voltage (LOV) domains that function as blue light sensors to control cellular responses to light. In the second LOV domain of phototropins, called LOV2 domains, blue light illumination leads to covalent bond formation between protein and flavin that induces the dissociation and unfolding of a C-terminally attached α helix (Jα) and the N-terminal helix (A′α). To date, the majority of studies on these domains have focused on versions that contain truncations in the termini, which creates difficulties when extrapolating to the much larger proteins that contain these domains. Here, we study the influence of deletions and extensions of the A′α helix of the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) on the light-triggered structural response of the protein by Fourier-transform infrared difference spectroscopy. Deletion of the A′α helix abolishes the light-induced unfolding of Jα, whereas extensions of the A′α helix lead to an attenuated structural change of Jα. These results are different from shorter constructs, indicating that the conformational changes in full-length phototropin LOV domains might not be as large as previously assumed, and that the well-characterized full unfolding of the Jα helix in AsLOV2 with short A′α helices may be considered a truncation artifact. It also suggests that the N- and C-terminal helices of phot-LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may provide a basis for signal integration of LOV1 and LOV2 domains in phototropins

Relative contributions of norspermidine synthesis and signaling pathways to the regulation of Vibrio cholerae biofilm formation.

The polyamine norspermidine is one of the major polyamines synthesized by Vibrionales and has also been found in various aquatic organisms. Norspermidine is among the environmental signals that positively regulate Vibrio cholerae biofilm formation. The NspS/MbaA signaling complex detects extracellular norspermidine and mediates the response to this polyamine. Norspermidine binding to the NspS periplasmic binding protein is thought to inhibit the phosphodiesterase activity of MbaA, increasing levels of the biofilm-promoting second messenger cyclic diguanylate monophosphate, thus enhancing biofilm formation. V. cholerae can also synthesize norspermidine using the enzyme NspC as well as import it from the environment. Deletion of the nspC gene was shown to reduce accumulation of bacteria in biofilms, leading to the conclusion that intracellular norspermidine is also a positive regulator of biofilm formation. Because V. cholerae uses norspermidine to synthesize the siderophore vibriobactin it is possible that intracellular norspermidine is required to obtain sufficient amounts of iron, which is also necessary for robust biofilm formation. The objective of this study was to assess the relative contributions of intracellular and extracellular norspermidine to the regulation of biofilm formation in V. cholerae. We show the biofilm defect of norspermidine synthesis mutants does not result from an inability to produce vibriobactin as vibriobactin synthesis mutants do not have diminished biofilm forming abilities. Furthermore, our work shows that extracellular, but not intracellular norspermidine, is mainly responsible for promoting biofilm formation. We establish that the NspS/MbaA signaling complex is the dominant mediator of biofilm formation in response to extracellular norspermidine, rather than norspermidine synthesized by NspC or imported into the cell