| Cryptochrome-1 | |||||||
|---|---|---|---|---|---|---|---|
 Crystallographic structure of Cryptochrome-1 | |||||||
| Identifiers | |||||||
| Symbol | CRY1 | ||||||
| NCBI gene | 1407 | ||||||
| HGNC | 2384 | ||||||
| OMIM | 601933 | ||||||
| PDB | 5T5X | ||||||
| RefSeq | NP_004066 | ||||||
| UniProt | Q16526 | ||||||
| Other data | |||||||
| Locus | Chr. 12q23.3 | ||||||
| |||||||
| Cryptochrome-2 | |||||||
|---|---|---|---|---|---|---|---|
| Identifiers | |||||||
| Symbol | CRY2 | ||||||
| NCBI gene | 1408 | ||||||
| HGNC | 2385 | ||||||
| OMIM | 603732 | ||||||
| PDB | 4MLP | ||||||
| RefSeq | NP_066940 | ||||||
| UniProt | Q49AN0 | ||||||
| Other data | |||||||
| Locus | Chr. 11p11.2 | ||||||
| |||||||
Cryptochromes (from theGreek κρυπτός χρώμα, "hidden colour") are a class offlavoproteins found inplants andanimals that are sensitive toblue light. They are involved in thecircadian rhythms and thesensing of magnetic fields in a number of species. The namecryptochrome was proposed as aportmanteau combining thechromatic nature of thephotoreceptor, and thecryptogamic organisms on which many blue-light studies were carried out.[1][2]
ThegenesCRY1 andCRY2 encode theproteins CRY1 and CRY2, respectively.[3] Cryptochromes are classified into plant Cry and animal Cry. Animal Cry can be further categorized into insect type (Type I) and mammal-like (Type II). CRY1 is a circadianphotoreceptor whereas CRY2 is a clockrepressor which represses Clock/Cycle (Bmal1) complex in insects andvertebrates.[4] In plants, blue-light photoreception can be used to cue developmental signals.[5] Besideschlorophylls, cryptochromes are the only proteins known to form photoinducedradical-pairsin vivo.[6] These appear to enable some animals to detect magnetic fields.
Cryptochromes have been the focus of several efforts inoptogenetics. Employingtransfection, initial studies on yeast have capitalized on the potential of CRY2heterodimerization to control cellular processes, includinggene expression, by light.
AlthoughCharles Darwin first documented plant responses to blue light in the 1880s, it was not until the 1980s that research began to identify the pigment responsible.[7] In 1980, researchers discovered that the HY4 gene of the plantArabidopsis thaliana was necessary for the plant's blue light sensitivity, and, when the gene was sequenced in 1993, it showed high sequence homology withphotolyase, a DNA repair protein activated by blue light.[8] Reference sequence analysis of cryptochrome-1 isoform d shows twoconserved domains with photolyase proteins. Isoform d nucleotide positions 6 through 491 show a conserved domain withdeoxyribodipyrimidine photolyase, and positions 288 through 486 show a conserved domain with the FAD binding domain of DNA photolyase.[9] Comparative genomic analysis supports photolyase proteins as the ancestors of cryptochromes. However, by 1995 it became clear that the products of the HY4 gene and its two humanhomologs did not exhibit photolyase activity and were instead a new class ofblue light photoreceptor hypothesized to becircadianphotopigments.[10] In 1996 and 1998,Cry homologs were identified inDrosophila andmice, respectively.[11][12]
Cryptochromes (CRY1, CRY2) are evolutionarily old and highly conserved proteins that belong to the flavoproteins superfamily that exists in all kingdoms of life. Cryptochromes are derived from and closely related to photolyases, which are bacterialenzymes that are activated by light and involved in the repair of UV-inducedDNA damage.
Ineukaryotes, cryptochromes no longer retain this original enzymatic activity. By using aT-DNA labeled allele of thecry1 gene in theArabidopsis plant, researchers determined that thecry1 gene encoded a flavoprotein without photolyase activity and with a uniqueC-terminal tail.[13] The protein encoded by this gene was named cryptochrome 1 to distinguish it from its ancestral photolyase proteins and was found to be involved in the photoreception of blue light. Studies ofDrosophila cry-knockout mutants led to the later discovery that cryptochrome proteins are also involved in regulating the mammalian circadian clock. TheDrosophila cry gene similarly encodes a flavoprotein without photolyase activity that also bindspterinchromophores.[13]Cry mutants (cryb) were found to express arrhythmic levels ofluciferase as well asPER andTIM proteins in photoreceptor cells.[13] Despite the arrhythmicity of these protein levels,cryb mutants still showed rhythmicity in overall behavior but could notentrain to short pulses of light, leading researchers to conclude that the dorsal and ventral lateral neurons (the primarypacemaker cells ofDrosophila) were still functioning effectively.[13] Whencryb mutants also had visually unresponsive compound eyes, though, they failed to behaviorally entrain toenvironmental cues.[13] These findings led researchers to conclude that the cryptochrome protein encoded bycry is necessary forDrosophila photoentrainment. In mammals, a protein analog of theDrosophila cryptochrome protein was discovered with the characteristic property of lacking photolyase activity, prompting researchers to consider it in the same class of cryptochrome proteins.[13] In mice, the greatestcry1 expression is observed in thesuprachiasmatic nucleus (SCN) where levels rhythmically fluctuate.[13] Due to the role of the SCN as the primary mammalian pacemaker as well as the rhythmic fluctuations incry1 expression, researchers concludedcry1 was also necessary for the entrainment of mammalian circadian rhythms.
A common misconception in the evolutionary history of cryptochrome proteins is that mammalian and plant proteins areorthologs of each other that evolved directly from a shared photolyase gene. However, genomic analysis indicates that mammalian and fly cryptochrome proteins show greatersequence similarity to the (6-4) photolyase proteins than to plant cryptochrome proteins.[13] It is therefore likely that plant and animal cryptochrome proteins show a unique case ofconvergent evolution by repeatedly evolving new functions independently of each other from a single common ancestralcry gene.[13]
Research by Worthington et al. (2003) indicates that cryptochromes first evolved in bacteria and were identified inVibrio cholerae.[14] Genome sequencing of this bacteria identified three genes in the photolyase/cryptochrome family, all of which have thefolate andflavin cofactors characteristic of these proteins.[14] Of these genes, one encodes a photolyase, while the other two encode cryptochrome proteins designated VcCry1 and VcCry2.[14] Cashmore AR et al. (1999) hypothesize that mammalian cryptochromes developed later in evolutionary history shortly after plants and animals diverged based on conserved genomic domains between animal cryptochromes and theArabidopsis (6-4) photolyase protein.[13] Based on the role of cryptochromes in the entrainment of mammalian circadian rhythms, current researchers hypothesize that they developed simultaneously with the coevolution of PER, TIM,CLOCK, andCYCLE proteins, but there is currently insufficient evidence to determine the exact evolution timing and mechanism of evolution.[13]
All members of the flavoprotein superfamily have the characteristics of anN-terminalphotolyase homology (PHR) domain. The PHR domain can bind to theflavin adenine dinucleotide (FAD)cofactor and alight-harvestingchromophore.[15] The structure of cryptochrome involves a fold very similar to that of photolyase, arranged as an orthogonal bundle with a single molecule of FADnoncovalently bound to the protein.[15] These proteins have variable lengths and surfaces on the C-terminal end, due to the changes in genome and appearance that result from the lack ofDNA repair enzymes.[15] TheRamachandran plot shows that thesecondary structure of the CRY1 protein is primarily a right-handedalpha helix with little to no steric overlap. The structure of CRY1 is almost entirely made up of alpha helices, with several loops and fewbeta sheets.[15]
In plants, cryptochromes mediatephototropism, or directional growth toward a light source, in response to blue light. This response is now known to have its own set of photoreceptors, thephototropins.
Unlikephytochromes and phototropins, cryptochromes are notkinases. Theirflavinchromophore is reduced by light and transported into thecell nucleus, where it affects theturgor pressure and causes subsequent stem elongation. To be specific,Cry2 is responsible for blue-light-mediatedcotyledon and leaf expansion.Cry2 overexpression intransgenic plants increases blue-light-stimulated cotyledon expansion, which results in many broad leaves and no flowers rather than a few primary leaves with a flower.[16] A double loss-of-function mutation in Arabidopsis thaliana Early Flowering 3 (elf3) and Cry2 genes delays flowering under continuous light and was shown to accelerate it during long and short days, which suggests that Arabidopsis CRY2 may play a role in accelerating flowering time during continuous light.[17]
Cryptochromes receptors cause plants to respond to blue light viaphotomorphogenesis. They help control seed and seedling development, as well as the switch from the vegetative to the flowering stage of development.
InArabidopsis, CRY1 is the primary inhibitor of hypocotyl elongation but CRY2 inhibits hypocotyl elongation under low blue light intensity. CRY2 promotes flowering under long-day conditions.[18]
CRY gene mediates photomorphogenesis in several ways. CRY C-terminal interacts with CONTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a E3 ubiquitin ligase that represses photomorphogenesis and flowering time. The interaction inhibits COP1 activity and allows transcription factors such as ELONGATED HYPOCOTYL 5 (HY5) to accumulate.[19] HY5 is a basic leucine zipper (bZIP) factor that promotes photomorphogenesis by binding to light-responsive genes. CRY interacts with G protein β-subunit AGB1, where HY5 dissociates from AGB1 and becomes activated. CRY interacts with PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PIF5, repressors of photomorphogenesis and promoter of hypocotyl elongation, to repress PIF4 and PIF5 transcription activity. Lastly, CRY can inhibitauxin andbrassinosterioid (BR) signaling to promote photomorphogenesis.[18]
Despite much research on the topic, cryptochromephotoreception andphototransduction inDrosophila andArabidopsis thaliana is still poorly understood. Cryptochromes are known to possess two chromophores:pterin (in the form of5,10-methenyltetrahydrofolic acid (MTHF)) and flavin (in the form of FAD).[20] Both may absorb aphoton, and inArabidopsis, pterin appears to absorb at a wavelength of 380 nm and flavin at 450 nm. Past studies have supported a model by which energy captured by pterin is transferred to flavin.[21] Under this model of phototransduction, FAD would then bereduced to FADH, which probably mediates thephosphorylation of a certain domain in cryptochrome. This could then trigger asignal transduction chain, possibly affectinggene regulation in thecell nucleus.
A new hypothesis[22] proposes that partner molecules sense the transduction of a light signal into a chemical signal in plant cryptochromes, which could be triggered by a photo-induced negative charge on the FAD cofactor or on the neighboring aspartic acid[23][24] within the protein. This negative charge would electrostatically repel the protein-boundATP molecule and thereby also the protein C-terminal domain, which covers theATP binding pocket prior to photon absorption. The resulting change in protein conformation could lead to phosphorylation of previously inaccessible phosphorylation sites on the C-terminus and the given phosphorylated segment could then liberate the transcription factor HY5 by competing for the same binding site at the negative regulator of photomorphogenesisCOP1.
A different mechanism may function inDrosophila. The true ground state of the flavin cofactor inDrosophila CRY is still debated, with some models indicating that the FAD is in an oxidized form,[25] while others support a model in which the flavin cofactor exists inanionradical form,FAD−
•. Recently, researchers have observed that oxidized FAD is readily reduced toFAD−
• by light. Furthermore, mutations that blocked photoreduction had no effect on light-induced degradation of CRY, while mutations that altered the stability ofFAD−
• destroyed CRY photoreceptor function.[26][27] These observations provide support for a ground state ofFAD−
•. Researchers have also recently proposed a model in whichFAD−
is excited to itsdoublet or quartet state by absorption of a photon, which then leads to a conformational change in the CRY protein.[28]
Also the ring eyes of thedemosponge larva ofAmphimedon queenslandica express a blue-light-sensitive cryptochrome (Aq-Cry2), which might mediate phototaxis. In contrast, the eyes of most animals usephoto-sensitiveopsins expressed in photoreceptor cells, which communicate information about light from the environment to the nervous system. However,A. queenslandica lacks a nervous system, like othersponges. And it does not have anopsingene in its fully sequencedgenome either, despite having many otherG-protein-coupled receptors (GPCRs). Therefore, the sponge's unique eyes must have evolved a different mechanism to detect light and mediate phototaxis, possibly with cryptochromes or other proteins.[29]
Isolated irises constrict in response to light via a photomechanical transduction response (PMTR) in a variety of species and require eithermelanopsin or cryptochrome to do so.[30] The iris of chicken embryos senses short-wavelength light via a cryptochrome, rather than opsins.[31] Research by Margiotta and Howard (2020) shows that the PMTR of the chicken iris striated muscle occurs withCRY gene activation by 430 nm blue light.[30] The PMTR was inhibited inCRY gene knockouts and decreased when flavin reductase was inhibited, but remained intact with the addition of melanopsin antagonists.[30] Similarly, cytosolicCRY1 andCRY2 proteins were found in irismyotubes, and decreasing transcription of these genes inhibited PMTRs.[30] The greatest iris PMTRs therefore correspond with the development of striated, rather than smooth, muscle fibers throughCRY-mediated PMTRs.[30]
Studies in animals and plants suggest that cryptochromes play a pivotal role in the generation and maintenance of circadian rhythms.[32] Similarly, cryptochromes play an important role in the entrainment of circadian rhythms in plants.[33] InDrosophila, cryptochrome (dCRY) acts as a blue-light photoreceptor that directly modulates light input into the circadian clock,[34] while in mammals, cryptochromes (CRY1 and CRY2) act astranscriptionrepressors within the circadian clockwork.[35] Some insects, including themonarch butterfly, have both a mammal-like and aDrosophila-like version of cryptochrome, providing evidence for an ancestral clock mechanism involving both light-sensing and transcriptional-repression roles for cryptochrome.[36][37]
Crymutants have altered circadian rhythms, showing thatCry affects the circadian pacemaker.Drosophila with mutatedCry exhibit little to no mRNA cycling.[38] A point mutation incryb, which is required for flavin association in CRY protein, results in no PER or TIM protein cycling in either DD or LD.[39] In addition, mice lackingCry1 orCry2 genes exhibit differentially altered free running periods, but are still capable ofphotoentrainment. However, mice that lack bothCry1 andCry2 are arrhythmic in both LD and DD and always have highPer1 mRNA levels. These results suggest that cryptochromes play a photoreceptive role, as well as acting as negative regulators of Per gene expression in mice.[40]
InDrosophila, cryptochrome is only encoded by oneCry gene (dCry) and functions as a blue light photoreceptor. Exposure to blue light induces a conformation similar to that of the always-active CRY mutant with a C-terminal deletion (CRYΔ).[28] The half-life of this conformation is 15 minutes in the dark and facilitates the binding of CRY to other clock gene products, PER andTIM, in a light-dependent manner.[41][28][34][42] Once bound by dCRY, dTIM is committed to degradation by the ubiquitin-proteasome system.[28][42]
Although light pulses do not entrain, full photoperiod LD cycles can still drive cycling in theventral-lateral neurons in theDrosophila brain. These data along with other results suggest that CRY is the cell-autonomous photoreceptor for body clocks inDrosophila and may play a role in nonparametric entrainment (entrainment by short discrete light pulses). However, the lateral neurons receive light information through both the blue light CRY pathway and therhodopsin pathway. Therefore, CRY is involved in light perception and is an input to the circadian clock, however it is not the only input for light information, as a sustained rhythm has been shown in the absence of the CRY pathway, in which it is believed that the rhodopsin pathway is providing some light input.[43] Recently, it has also been shown that there is a CRY-mediated light response that is independent of the classical circadian CRY-TIM interaction. This mechanism is believed to require a flavinredox-based mechanism that is dependent on potassium channel conductance. This CRY-mediated light response has been shown to increaseaction potential firing within seconds of a light response inopsin-knockoutDrosophila.[44]
Cryptochrome, like many genes involved in circadian rhythm, shows circadian cycling inmRNA and protein levels. InDrosophila,Cry mRNA concentrations cycle under a light-dark cycle (LD), with high levels in light and low levels in the dark.[38] This cycling persists in constant darkness (DD), but with decreased amplitude.[38] The transcription of theCry gene also cycles with a similar trend.[38] CRY protein levels, however, cycle in a different manner thanCry transcription and mRNA levels. In LD, CRY protein has low levels in light and high levels in dark, and, in DD, CRY levels increase continuously throughout the subjective day and night.[38] Thus, CRY expression is regulated by the clock at the transcriptional level and by light at thetranslational and posttranslational level.[38]
Overexpression ofCry also affects circadian light responses. InDrosophila,Cry overexpression increases flies' sensitivity to low-intensity light.[38] This light regulation of CRY protein levels suggests that CRY has a circadian role upstream of other clock genes and components.[38]
In mammals, cryptochrome proteins are encoded by two genes,Cry1 andCry2.
Cryptochrome is one of the four groups of mammalian clock genes/proteins that generate a transcription-translation negative-feedback loop (TTFL), along withPeriod (PER),CLOCK, andBMAL1.[45] In this loop, CLOCK and BMAL1 proteins aretranscriptional activators, which together bind to thepromoters of theCry2 andPer genes and activate their transcription.[45] The CRY2 and PER proteins then bind to each other, enter the nucleus, and inhibit CLOCK-BMAL1-activated transcription.[45] The overall function of CRY2 is therefore to repress transcription of CLOCK and BMAL1.
Cry1 encodes the CRY1 protein which is a mammalian circadian photoreceptor. In mice,Cry1 expression displays circadian rhythms in thesuprachiasmatic nucleus, a brain region involved in the generation of circadian rhythms, with mRNA levels peaking during the light phase and reaching a minimum in the dark.[46] These daily oscillations in expression are maintained in constant darkness.[46]
While CRY1 has been well established as a TIM homolog in mammals, the role of CRY1 as a photoreceptor in mammals has been controversial. Early papers indicated that CRY1 has both light-independent and -dependent functions. A study conducted by Selby CP et al. (2000) found that mice without rhodopsin but with cryptochrome still respond to light; however, in mice without either rhodopsin or cryptochrome,c-Fos transcription, a mediator of light sensitivity, significantly drops.[47] In recent years, data have supportedmelanopsin as the main circadian photoreceptor, in particular melanopsin cells that mediate entrainment and communication between theeye and the suprachiasmatic nucleus (SCN).[48] One of the main difficulties in confirming or denying CRY as a mammalian photoreceptor is that when the gene is knocked out the animal goes arrhythmic, so it is hard to measure its capacity as purely a photoreceptor. However, some recent studies indicate that human CRY1 may mediate light response in peripheral tissues.[49]
Normal mammalian circadian rhythm relies critically on delayed expression ofCry1 following activation of theCry1 promoter. Whereas rhythms inPer2 promoter activation andPer2 mRNA levels have almost the same phase,Cry1 mRNA production is delayed by approximately four hours relative toCry1 promoter activation.[50] This delay is independent of CRY1 or CRY2 levels and is mediated by a combination ofE/E'-box and D-box elements in the promoter andRevErbA/ROR binding elements (RREs) in the gene's first intron.[51]Transfection of arrhythmicCry1−/−Cry2−/− double-knockout cells with only theCry1 promoter (causing constitutiveCry1 expression) is not sufficient to rescue rhythmicity. Transfection of these cells with both the promoter and the firstintron is required for restoration of circadian rhythms in these cells.[51]
There is evidence that CRY1 can play a role in how sleep-wake patterns can beinherited through families. There is a mutation,CRY1Δ11, that causes a delay in one's circadian rhythm.[52] CRY1Δ11 is a splicing variant that has deleted anauto-inhibitory section of the gene.[52] It causes a delay by increasing the affinity of CLOCK andBMAL which in turn lengthens the period.[52] This causes people with this mutation to have a later sleep midpoint than the rest of the population, causing a disorder known asdelayed sleep–wake phase disorder.[52]
CRY1 is also a key modulator inDNA repair, specifically through temporal regulation.[53] CRY1 has an impact in the cell cycle progression, particularly in theG2/M checkpoint, and thedepletion of CRY1 leads to effects on DNA repair networks, including mismatch repair, UV, andnucleotide excision.[53] Incancer, CRY1 is stabilized by DNA damage, which results in CRY1 expression being associated with worse outcomes inprostate cancer.[53] Because of its role in DNA repair and beingpro-tumorigenic, further research can use CRY1 as atherapeutic target.
Variants of CRY1 can have impacts on humans in regards to metabolic output. According to a 2021 study,metabolic outputs, measured bybowel movements, were severely different for participants who werewild type in comparison to those with the CRY1Δ11 variant.[52] The participants with the variant had a delayed sleep cycle anddelayed metabolic output when compared to the wild type.[52]
Magnetoreception is a sense which allows an organism to detect a magnetic field to perceive direction, altitude or location. Experimental data suggests that cryptochromes in thephotoreceptor neurons of birds' eyes are involved in magnetic orientation duringmigration.[55] Cryptochromes are also thought to be essential for the light-dependent ability ofDrosophila to sensemagnetic fields.[56] Magnetic fields were once reported to affect cryptochromes also inArabidopsis thaliana plants: growth behavior seemed to be affected by magnetic fields in the presence of blue (but not red) light.[57] Nevertheless, these results have later turned out to be irreproducible under strictly controlled conditions in another laboratory,[58] suggesting that plant cryptochromes do not respond to magnetic fields.
Cryptochrome forms a pair ofradicals with correlatedspins when exposed to blue light.[59][60] Radical pairs can also be generated by the light-independent dark reoxidation of the flavin cofactor by molecular oxygen through the formation of a spin-correlated FADH-superoxide radical pairs.[61] Magnetoreception is hypothesized to function through the surrounding magnetic field's effect on the correlation (parallel or anti-parallel) of these radicals, which affects the lifetime of the activated form of cryptochrome. Activation of cryptochrome may affect the light-sensitivity ofretinal neurons, with the overall result that the animal can sense the magnetic field.[62] Animal cryptochromes and closely related animal (6-4) photolyases contain a longer chain of electron-transferring tryptophans than other proteins of the cryptochrome-photolyase superfamily (a tryptophan tetrad instead of a triad).[63][64] The longer chain leads to a better separation and over 1000× longer lifetimes of the photoinduced flavin-tryptophan radical pairs than in proteins with a triad of tryptophans.[63][64] The absence of spin-selective recombination of these radical pairs on the nanosecond to microsecond timescales seems to be incompatible with the suggestion that magnetoreception by cryptochromes is based on the forward light reaction.