Browse
Submit Data
Databases
API
Help

Dataset Information

10 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Human and Drosophila cryptochromes are light activated by flavin photoreduction in living cells.

ABSTRACT: Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non-light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.

SUBMITTER: Hoang N

PROVIDER: S-EPMC2443192 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Peripheral Methionine Residues Impact Flavin Photoreduction and Protonation in an Engineered LOV Domain Light Sensor.

Project description:Proton-coupled electron transfer reactions play critical roles in many aspects of sensory phototransduction. In the case of flavoprotein light sensors, reductive quenching of flavin excited states initiates chemical and conformational changes that ultimately transmit light signals to downstream targets. These reactions generally require neighboring aromatic residues and proton-donating side chains for rapid and coordinated electron and proton transfer to flavin. Although photoreduction of flavoproteins can produce either the anionic (ASQ) or neutral semiquinone (NSQ), the factors that favor one over the other are not well understood. Here we employ a biologically active variant of the light-oxygen-voltage (LOV) domain protein VVD devoid of the adduct-forming Cys residue (VVD-III) to probe the mechanism of flavin photoreduction and protonation. A series of isosteric and conservative residue replacements studied by rate measurements, fluorescence quantum yields, FTIR difference spectroscopy, and molecular dynamics simulations indicate that tyrosine residues facilitate charge recombination reactions that limit sustained flavin reduction, whereas methionine residues facilitate radical propagation and quenching and also gate solvent access for flavin protonation. Replacement of a single surface Met residue with Leu favors formation of the ASQ over the NSQ and desensitizes photoreduction to oxidants. In contrast, increasing site hydrophilicity by Gln substitution promotes rapid NSQ formation and weakens the influence of the redox environment. Overall, the photoreactivity of VVD-III can be understood in terms of redundant electron donors, internal hole quenching, and coupled proton transfer reactions that all depend upon protein conformation, dynamics, and solvent penetration.

| S-EPMC8107827 | biostudies-literature

Human cryptochrome-1 confers light independent biological activity in transgenic Drosophila correlated with flavin radical stability.

Project description:Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome--1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism.

| S-EPMC3299647 | biostudies-literature

Light-activated kinases enable temporal dissection of signaling networks in living cells.

Project description:We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway.

| S-EPMC3048767 | biostudies-literature

The structural and functional roles of the flavin cofactor FAD in mammalian cryptochromes.

Project description:The importance of circadian rhythms in human health and disease calls for a thorough understanding of the underlying molecular machinery, including its key components, the flavin adenine dinucleotide (FAD)-containing flavoproteins cryptochrome 1 and 2. Contrary to their Drosophila counterparts, mammalian cryptochromes are direct suppressors of circadian transcription and act independently of light. Light-independence poses the question regarding the role of the cofactor FAD in mammalian cryptochromes. The weak binding of the cofactor in vitro argues against its relevance and might be a functionless evolutionary remnant. From the other side, the FAD-binding pocket constitutes the part of mammalian cryptochromes directly related to their ubiquitylation by the ubiquitin ligase Fbxl3 and is the target for protein-stabilizing small molecules. Increased supplies of FAD stabilize cryptochromes in cell culture, and the depletion of the FAD precursor riboflavin with simultaneous knock-down of riboflavin kinase affects the expression of circadian genes in mice. This review presents the classical and more recent studies in the field, which help to comprehend the role of FAD for the stability and function of mammalian cryptochromes.

| S-EPMC9845712 | biostudies-literature

Light-Activated Staudinger-Bertozzi Ligation within Living Animals.

Project description:The ability to regulate small molecule chemistry in vivo will enable new avenues of exploration in imaging and pharmacology. However, realization of these goals will require reactions with high specificity and precise control. Here we demonstrate photocontrol over the highly specific Staudinger-Bertozzi ligation in vitro and in vivo. Our simple approach, photocaging the key phosphine atom, allows for the facile production of reagents with photochemistry that can be engineered for specific applications. The resulting compounds, which are both stable and efficiently activated, enable the spatial labeling of metabolically introduced azides in vitro and on live zebrafish.

| S-EPMC5174980 | biostudies-literature

Tuning flavin environment to detect and control light-induced conformational switching in Drosophila cryptochrome.

Project description:Light-induction of an anionic semiquinone (SQ) flavin radical in Drosophila cryptochrome (dCRY) alters the dCRY conformation to promote binding and degradation of the circadian clock protein Timeless (TIM). Specific peptide ligation with sortase A attaches a nitroxide spin-probe to the dCRY C-terminal tail (CTT) while avoiding deleterious side reactions. Pulse dipolar electron-spin resonance spectroscopy from the CTT nitroxide to the SQ shows that flavin photoreduction shifts the CTT ~1 nm and increases its motion, without causing full displacement from the protein. dCRY engineered to form the neutral SQ serves as a dark-state proxy to reveal that the CTT remains docked when the flavin ring is reduced but uncharged. Substitutions of flavin-proximal His378 promote CTT undocking in the dark or diminish undocking in the light, consistent with molecular dynamics simulations and TIM degradation activity. The His378 variants inform on recognition motifs for dCRY cellular turnover and strategies for developing optogenetic tools.

| S-EPMC7910608 | biostudies-literature

Cryptochromes integrate green light signals into the circadian system.

Project description:Plants are acutely sensitive of their light environment, adapting their growth habit and prioritizing developmental decisions to maximize fecundity. In addition to providing an energy source and directional information, light quality also contributes to entrainment of the circadian system, an endogenous timing mechanism that integrates endogenous and environmental signalling cues to promote growth. Whereas plants' perception of red and blue portions of the spectrum are well defined, green light sensitivity remains enigmatic. In this study, we show that low fluence rates of green light are sufficient to entrain and maintain circadian rhythms in Arabidopsis and that cryptochromes contribute to this response. Importantly, green light responses are distinguishable from low blue light-induced phenotypes. These data suggest a distinct signalling mechanism enables entrainment of the circadian system in green light-enriched environments, such as those found in undergrowth and in densely planted monoculture.

| S-EPMC6973147 | biostudies-literature

Regulating gene expression in human leukemia cells using light-activated oligodeoxynucleotides.

Project description:Light-activated antisense oligodeoxynucleotides (asODNs) were developed to control the degradation of target mRNA in living cells by RNase H. A 20-mer asODN previously shown to target c-myb, a hematopoietic transcription factor, was covalently attached via a photocleavable linker (PL) to partially complementary 20-mer sense strands (sODNs). In the 'caged' state, the sODN blocked hybridization of the asODN to c-myb mRNA. Six asODN-PL-sODN conjugates, C1-C6, were synthesized. C5, with twelve complementary bases, gave the largest decrease in melting temperature (T(m)) upon UV irradiation (DeltaT(m) = -29 degrees C). The most thermally stable conjugate, C6 (T(m) = 84 degrees C), gave the lowest background RNase H activity, with just 8.6% degradation of an RNA 40-mer after 1 h incubation. In biochemical assays with C6, RNA digestion increased 10-fold 10 min after UV irradiation. Finally, phosphorothioated analogs S-C5 and S-C6 were synthesized to test activity in cultured K562 (human leukemia) cells. No knockdown of c-myb mRNA or protein was observed with intact S-C5 or S-C6, whereas more than half of c-myb mRNA was degraded 24 h after photoactivation. Two-fold photomodulation of c-MYB protein levels was also observed with S-C5. However, no photomodulation of c-MYB protein levels was observed with S-C6, perhaps due to the greater stability of this duplex.

| S-EPMC2241881 | biostudies-literature

Flavin reduction activates Drosophila cryptochrome.

Project description:Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation.

| S-EPMC3870761 | biostudies-literature

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins.

Project description:Single-molecule (SM) microscopy allows outstanding insight into biomolecular mechanisms in cells. However, selective detection of single biomolecules in their native environment remains particularly challenging. Here, we introduce an easy methodology that combines specific targeting and nanometer accuracy imaging of individual biomolecules in living cells. In this method, named complementation-activated light microscopy (CALM), proteins are fused to dark split-fluorescent proteins (split-FPs), which are activated into bright FPs by complementation with synthetic peptides. Using CALM, the diffusion dynamics of a controlled subset of extracellular and intracellular proteins are imaged with nanometer precision, and SM tracking can additionally be performed with fluorophores and quantum dots. In cells, site-specific labeling of these probes is verified by coincidence SM detection with the complemented split-FP fusion proteins or intramolecular single-pair Förster resonance energy transfer. CALM is simple and combines advantages from genetically encoded and synthetic fluorescent probes to allow high-accuracy imaging of single biomolecules in living cells, independently of their expression level and at very high probe concentrations.

| S-EPMC3116416 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data