Showing 1 - 12 of 12 results
Light-controllable Transcription System by Nucleocytoplasmic Shuttling of a Truncated Phytochrome B.
Transcriptional regulation is a useful strategy for gene therapy and for biomedical research. Unlike chemically regulated transcriptional approaches, spatiotemporal control of transcription using optogenetic tools is a powerful technology for the analysis of single cells. For light to penetrate into tissues, it is desired to use photoreceptors absorbing red/far-red light with a low-molecular mass applicable for the use of virus vectors, and a photoswitch using the photoreceptor need to be constructed as a single expression vector. Herein, we describe an optogenetic tool based on Arabidopsis thaliana phytochrome (Phy) B and its binding partner, phytochrome-interacting factor (PIF) 6. We generated a truncated PhyB, which allowed for reversible association with PIF6 by red/far-red light illumination. The red light illumination only for 5 min induced PhyB translocation from cytoplasm into the nucleus by the association with PIF6, resulting in transcriptional activation based on Gal4 DNA-binding domain and the upstream activating sequence of Gal system. The nucleocytoplasmic shuttling vector using PhyB and PIF6 might be applicable for transcriptional regulation in tissue experiments. This article is protected by copyright. All rights reserved.
Interactions Between phyB and PIF Proteins Alter Thermal Reversion Reactions in vitro.
The dynamic behavior of the plant red/far-red light photoreceptor phytochrome B (phyB) has been elucidated in natural and synthetic systems. Red light switches phyB from the inactive Pr state to the active Pfr state, a process that is reversed by far-red light. Alongside light signals, phyB activity is constrained by thermal reversion (that is prominent in the dark) and protein-protein interactions between phyB, other phytochrome molecules, and, among others, PHYTOCHROME INTERACTING FACTORs (PIFs). Requirements for phyB-PIF association have been well studied and are central to light-regulated synthetic tools. However, it is unknown whether PIF interactions influence transitions of phyB between different conformers. Here, we show that the in vitro thermal reversion of phyB involves multiple reactions. Thermal reversion of phyB in vitro is inhibited by PIF6, and this effect is observed at all temperatures tested. We analyzed our experimental data using a mathematical model containing multiple Pfr conformers, in accordance with previous findings. Remarkably, each Pfr conformer is differentially regulated by PIF6 and temperature. As a result, we speculate that in vivo phytochrome signaling networks may require similar levels of complexity to fine-tune responses to the external environment.
Distinctive Properties of Dark Reversion Kinetics between Two Red/Green-Type Cyanobacteriochromes and their Application in the Photoregulation of cAMP Synthesis.
Cyanobacteriochromes (CBCRs) are photoreceptors that bind to a linear tetrapyrrole within a conserved cGMP-phosphodiesterase/adenylate cyclase/FhlA (GAF) domain and exhibit reversible photoconversion. Red/green-type CBCR GAF domains that photoconvert between red- (Pr) and green-absorbing (Pg) forms occur widely in various cyanobacteria. A putative phototaxis regulator, AnPixJ, contains multiple red/green-type CBCR GAF domains. We previously reported that AnPixJ's second domain (AnPixJg2) but not its fourth domain (AnPixJg4) shows red/green reversible photoconversion. Herein, we found that AnPixJg4 showed Pr-to-Pg photoconversion and rapid Pg-to-Pr dark reversion, whereas AnPixJg2 showed a barely detectable dark reversion. Site-directed mutagenesis revealed the involvement of six residues in Pg stability. Replacement at the Leu294/Ile660 positions of AnPixJg2/AnPixJg4 showed the highest influence on dark reversion kinetics. AnPixJg2_DR6, wherein the six residues of AnPixJg2 were entirely replaced with those of AnPixJg4, showed a 300-fold faster dark reversion than that of the wild type. We constructed chimeric proteins by fusing the GAF domains with adenylate cyclase catalytic regions, such as AnPixJg2-AC, AnPixJg4-AC and AnPixJg2_DR6-AC. We detected successful enzymatic activation under red light for both AnPixJg2-AC and AnPixJg2_DR6-AC, and repression under green light for AnPixJg2-AC and under dark incubation for AnPixJg2_DR6-AC. These results provide platforms to develop cAMP synthetic optogenetic tools.
Strategies for development of optogenetic systems and their applications.
It has become clear that biological processes are highly dynamic and heterogeneous within and among cells. Conventional analytical tools and chemical or genetic manipulations are unsuitable for dissecting the role of their spatiotemporally dynamic nature. Recently, optical control of biomolecular signaling, a technology called “optogenetics,” has gained much attention. The technique has enabled spatial and temporal regulation of specific signaling pathways both in vitro and in vivo. This review presents strategies for optogenetic systems development and application for biological research. Combinations with other technologies and future perspectives are also discussed herein. Although many optogenetic approaches are designed to modulate ion channel conductivity, we mainly examine systems that target other biomolecular reactions such as gene expression, protein translocations, and kinase or receptor signaling pathways.
Optimizing optogenetic constructs for control over signaling and cell behaviours.
Optogenetic tools have recently been developed that enable dynamic control over the activities of select signaling proteins. They provide the unique ability to rapidly turn signaling events on or off with subcellular control in living cells and organisms. This capability is leading to new insights into how the spatial and temporal coordination of signaling events governs dynamic cell behaviours such as migration and neurite outgrowth. These tools can also be used to dissect a protein's signaling functions at different organelles. Here we review the properties of photoreceptors from diverse organisms that have been leveraged to control signaling in mammalian cells. We emphasize recent engineering approaches that have been used to create optogenetic constructs with optimized spectral, kinetic, and signaling properties for controlling cell behaviours.
How Does Photoreceptor UVR8 Perceive a UV-B Signal?
UVR8 is the only known plant photoreceptor that mediates light responses to UV-B (280-315 nm) of the solar spectrum. UVR8 perceives a UV-B signal via light-induced dimer dissociation, which triggers a wide range of cellular responses involved in photomorphogenesis and photoprotection. Two recent crystal structures of Arabidopsis thaliana UVR8 (AtUVR8) have revealed unusual clustering of UV-B-absorbing Trp pigments at the dimer interface and provided a structural framework for further mechanistic investigation. This review summarizes recent advances in spectroscopic, computational and crystallographic studies on UVR8 that are directed toward full understanding of UV-B perception at the molecular level.
Photo-dynamics of BLUF domain containing adenylyl cyclase NgPAC3 from the amoeboflagellate Naegleria gruberi NEG-M strain.
The absorption and emission spectroscopic behavior of the photo-activated adenylyl cyclase NgPAC3 from the amoeboflagellate Naegleria gruberi NEG-M strain was studied. The flavin cofactor was found to be partly fully oxidized and partly fully reduced. The typical BLUF domain (BLUF = Blue Light sensor Using Flavin) oxidized flavin absorption photo-cycle dynamics with about 14 nm flavin absorption red-shift in the signaling state was observed. The quantum efficiency of signaling state formation was determined to be s = 0.66 ± 0.03. A bi-exponential signaling state recovery to the dark-adapted receptor state was observed with the time constants rec,f = 275 s and rec,sl = 45 min. The thermal irreversible protein unfolding was studied and an apparent protein melting temperature of ϑm ≈ 50 ◦C was found. The photodynamic behavior of NgPAC3 is compared with the behavior of the previously investigated photo-activated cyclases NgPAC1 (nPAC) and NgPAC2 from the same N. gruberi NEG-M strain. Purified recombinant NgPAC3 showed light-gated adenylate cyclase activity upon illumination with blue light. Its cyclase activity is compared with those of NgPAC1 and NgPAC2.
Photoinduced damage to cellular DNA: direct and photosensitized reactions.
The survey focuses on recent aspects of photochemical reactions to cellular DNA that are implicated through the predominant formation of mostly bipyrimidine photoproducts in deleterious effects of human exposure to sunlight. Recent developments in analytical methods have allowed accurate and quantitative measurements of the main DNA photoproducts in cells and human skin. Highly mutagenic CC and CT bipyrimidine photoproducts, including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are generated in low yields with respect to TT and TC photoproducts. Another striking finding deals with the formation of Dewar valence isomers, the third class of bipyrimidine photoproducts that is accounted for by UVA-mediated isomerization of initially UVB generated 6-4PPs. Cyclobutadithymine (T<>T) has been unambiguously shown to be involved in the genotoxicity of UVA radiation. Thus, T<>T is formed in UVA-irradiated cellular DNA according to a direct excitation mechanism with a higher efficiency than oxidatively generated DNA damage that arises mostly through the Type II photosensitization mechanism. C<>C and C<>T are repaired at rates intermediate between those of T<>T and 6-4TT. Evidence has been also provided for the occurrence of photosensitized reactions mediated by exogenous agents that act either in an independent way or through photodynamic effects.
Old chromophores, new photoactivation paradigms, trendy applications: flavins in blue light-sensing photoreceptors.
The knowledge on the mechanisms by which blue light (BL) is sensed by diverse and numerous organisms, and of the physiological responses elicited by the BL photoreceptors, has grown remarkably during the last two decades. The basis for this "blue revival" was set by the identification and molecular characterization of long sought plant BL sensors, employing flavins as chromophores, chiefly cryptochromes and phototropins. The latter photosensors are the foundation members of the so-called light, oxygen, voltage (LOV)-protein family, largely spread among archaea, bacteria, fungi and plants. The accumulation of sequenced microbial genomes during the last years has added the BLUF (Blue Light sensing Using FAD) family to the BL photoreceptors and yielded the opportunity for intense "genome mining," which has presented to us the intriguing wealth of BL sensing in prokaryotes. In this contribution we provide an update of flavin-based BL sensors of the LOV and BLUF type, from prokaryotic microorganisms, with special emphasis to their light-activation pathways and molecular signal-transduction mechanisms. Rather than being a fully comprehensive review, this research collects the most recent discoveries and aims to unveil and compare signaling pathways and mechanisms of BL sensors.
A photoswitchable DNA-binding protein based on a truncated GCN4-photoactive yellow protein chimera.
Photo-controlled DNA-binding proteins promise to be useful tools for probing complex spatiotemporal patterns of gene expression in living organisms. Here we report a novel photoswitchable DNA-binding protein, GCN4(S)Δ25PYP, based on a truncated GCN4-photoactive yellow protein chimera. In contrast to previously reported designed photoswitchable proteins where DNA binding affinity is enhanced upon irradiation, GCN4(S)Δ25PYP dissociates from DNA when irradiated with blue light. In addition, the rate of thermal relaxation to the ground state, part of the PYP photocycle, is enhanced by DNA binding whereas in previous reported constructs it is slowed. The origins of this reversed photoactivity are analyzed in structural terms.
Structure and photoreaction of photoactive yellow protein, a structural prototype of the PAS domain superfamily.
Photoactive yellow protein (PYP) is a water-soluble photosensor protein found in purple photosynthetic bacteria. Unlike bacterial rhodopsins, photosensor proteins composed of seven transmembrane helices and a retinal chromophore in halophilic archaebacteria, PYP is a highly soluble globular protein. The alpha/beta fold structure of PYP is a structural prototype of the PAS domain superfamily, many members of which function as sensors for various kinds of stimuli. To absorb a photon in the visible region, PYP has a p-coumaric acid chromophore binding to the cysteine residue via a thioester bond. It exists in a deprotonated trans form in the dark. The primary photochemical event is photo-isomerization of the chromophore from trans to cis form. The twisted cis chromophore in early intermediates is relaxed and finally protonated. Consequently, the chromophore becomes electrostatically neutral and rearrangement of the hydrogen-bonding network triggers overall structural change of the protein moiety, in which local conformational change around the chromophore is propagated to the N-terminal region. Thus, it is an ideal model for protein conformational changes that result in functional change, responding to stimuli and expressing physiological activity. In this paper, recent progress in investigation of the photoresponse of PYP is reviewed.
Kinetic analysis of the activation of photoactivated adenylyl cyclase (PAC), a blue-light receptor for photomovements of Euglena.
Photoactivated adenylyl cyclase (PAC) was first purified from a photosensing organelle (the paraflagellar body) of the unicellular flagellate Euglena gracilis, and is regarded as the photoreceptor for the step-up photophobic response. Here, we report the kinetic properties of photoactivation of PAC and a change in intracellular cAMP levels upon blue light irradiation. Activation of PAC was dependent both on photon fluence rate and duration of irradiation, between which reciprocity held well in the range of 2--50 micromol m(-2) s(-1)(total fluence of 1200 micromol m(-2)). Intermittent irradiation also caused activation of PAC in a photon fluence-dependent manner irrespective of cycle periods. Wavelength dependency of PAC activation showed prominent peaks in the UV-B/C, UV-A and blue regions of the spectrum. The time course of the changes in intracellular cAMP levels corresponded well with that of the step-up photophobic response. From this and the kinetic properties of PAC photoactivation, we concluded that an increase in intracellular cAMP levels evoked by photoactivation of PAC is a key event of the step-up photophobic response.