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1.
Nat Plants ; 7(10): 1397-1408, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34650267

RESUMO

Cryptochromes (CRYs) are photoreceptors that mediate light regulation of the circadian clock in plants and animals. Here we show that CRYs mediate blue-light regulation of N6-methyladenosine (m6A) modification of more than 10% of messenger RNAs in the Arabidopsis transcriptome, especially those regulated by the circadian clock. CRY2 interacts with three subunits of the METTL3/14-type N6-methyladenosine RNA methyltransferase (m6A writer): MTA, MTB and FIP37. Photo-excited CRY2 undergoes liquid-liquid phase separation (LLPS) to co-condense m6A writer proteins in vivo, without obviously altering the affinity between CRY2 and the writer proteins. mta and cry1cry2 mutants share common defects of a lengthened circadian period, reduced m6A RNA methylation and accelerated degradation of mRNA encoding the core component of the molecular oscillator circadian clock associated 1 (CCA1). These results argue for a photoregulatory mechanism by which light-induced phase separation of CRYs modulates m6A writer activity, mRNA methylation and abundance, and the circadian rhythms in plants.


Assuntos
Adenosina/análogos & derivados , Arabidopsis/genética , Relógios Circadianos/genética , Criptocromos/metabolismo , Fotorreceptores de Plantas/metabolismo , Adenosina/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação
2.
Nat Commun ; 12(1): 5434, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521845

RESUMO

Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion. We find that vesicles mainly undergo kiss-and-run instead of full fusion in the absence of functional exocyst. Full fusion is rescued by optogenetically restoring exocyst function, in a manner likely dependent on the stoichiometry of tether engagement with the plasma membrane. In contrast, a passive artificial tether produces mostly kissing events, suggesting that kiss-and-run is the default mode of vesicle fusion. Optogenetic control of tethering further shows that fusion mode has physiological relevance since only full fusion could trigger lamellipodial expansion. These findings demonstrate that active coupling between tethering and fusion is critical for robust membrane merger.


Assuntos
Criptocromos/genética , Exossomos/metabolismo , Receptores da Transferrina/genética , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Criptocromos/metabolismo , Exossomos/ultraestrutura , Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fusão de Membrana/genética , Microscopia de Fluorescência , Optogenética/métodos , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/ultraestrutura , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
3.
Sci Rep ; 11(1): 18510, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531414

RESUMO

Circadian rhythm is an important mechanism that controls behavior and biochemical events based on 24 h rhythmicity. Ample evidence indicates disturbance of this mechanism is associated with different diseases such as cancer, mood disorders, and familial delayed phase sleep disorder. Therefore, drug discovery studies have been initiated using high throughput screening. Recently the crystal structures of core clock proteins (CLOCK/BMAL1, Cryptochromes (CRY), Periods), responsible for generating circadian rhythm, have been solved. Availability of structures makes amenable core clock proteins to design molecules regulating their activity by using in silico approaches. In addition to that, the implementation of classification features of molecules based on their toxicity and activity will improve the accuracy of the drug discovery process. Here, we identified 171 molecules that target functional domains of a core clock protein, CRY1, using structure-based drug design methods. We experimentally determined that 115 molecules were nontoxic, and 21 molecules significantly lengthened the period of circadian rhythm in U2OS cells. We then performed a machine learning study to classify these molecules for identifying features that make them toxic and lengthen the circadian period. Decision tree classifiers (DTC) identified 13 molecular descriptors, which predict the toxicity of molecules with a mean accuracy of 79.53% using tenfold cross-validation. Gradient boosting classifiers (XGBC) identified 10 molecular descriptors that predict and increase in the circadian period length with a mean accuracy of 86.56% with tenfold cross-validation. Our results suggested that these features can be used in QSAR studies to design novel nontoxic molecules that exhibit period lengthening activity.


Assuntos
Proteínas CLOCK/metabolismo , Ritmo Circadiano/fisiologia , Criptocromos/metabolismo , Animais , Bases de Dados de Proteínas , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica
4.
Life Sci ; 285: 119951, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34516994

RESUMO

AIMS: We sought to evaluate the effects of overfeeding during lactation on the feeding behavior and expression of specific regulatory genes in brain areas associated with food intake in 22- and 60-day old male rats. METHODS: We evaluated body weight, food intake of standard and palatable diet, and mRNA expression of dopamine receptor D1 (DDR1), dopamine receptor (DDR2), melanocortin 4 receptor (MC4R), the µ-opioid receptor (MOR), neuropeptide Y (NPY), agouti-related protein (AGRP), proopiomelanocortin (POMC), cocaine-and amphetamine-regulated transcript (CART), serotonin (5-hydroxytryptamine; 5-HT) transporter (SERT), 5-hydroxytryptamine receptor 1B (5-HT1B), 5-hydroxytryptamine receptor 2C receptor (5-HT2C), Clock (CLOK), cryptochrome protein 1 (Cry1) and period circadian protein homolog 2 (Per2) in the striatum, hypothalamus and brainstem of male rats at post-natal days (PND) 22 and 60. KEY FINDINGS: Overfeeding resulted in significantly increased body weight through PND60, and a 2-fold increase in palatable food intake at PND22, but not at PND60. We observed significant increases in DDR1, DDR2, and MC4R gene expression in the striatum and brainstem and POMC/CART in the hypothalamus of the OF group at PND22 that were reversed by PND60. Hypothalamic levels of 5-HT1B, 5-HT2C and NPY/AGRP on the other hand were decreased at PND22 and increased at PND60 in OF animals. Clock genes were unaffected by OF at PND22, but were significantly elevated at PND60. SIGNIFICANCE: Overfeeding during early development of the rat brain results in obesity and altered feeding behavior in early adulthood. The altered behavior might be the consequence of the changes in food intake and reward gene expression.


Assuntos
Peso Corporal , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiopatologia , Comportamento Alimentar , Hipernutrição/fisiopatologia , Animais , Proteínas CLOCK/metabolismo , Criptocromos/metabolismo , Ingestão de Alimentos , Feminino , Lactação , Masculino , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Wistar , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo
5.
J Biol Chem ; 297(3): 101068, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34375638

RESUMO

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.


Assuntos
Carcinogênese/efeitos dos fármacos , Cronofarmacocinética , Relógios Circadianos/fisiologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Proteínas CLOCK/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Ciclo Celular/fisiologia , Fenômenos Cronobiológicos , Relógios Circadianos/genética , Ritmo Circadiano/fisiologia , Cisplatino/farmacocinética , Cisplatino/farmacologia , Criptocromos/genética , Criptocromos/metabolismo , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/genética , Transcrição Genética/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Kidney Int ; 100(5): 1071-1080, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34332958

RESUMO

Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1 and 2 (Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietin mRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5' erythropoietin promoter. RNAscope® in situ hybridization confirmed the presence of Bmal1 in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy.


Assuntos
Relógios Circadianos , Eritropoetina , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Criptocromos/genética , Criptocromos/metabolismo , Regulação da Expressão Gênica , Homozigoto , Rim/metabolismo , Camundongos , Camundongos Knockout , Deleção de Sequência
7.
J Am Heart Assoc ; 10(16): e020896, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34348468

RESUMO

Background Circadian rhythm disorders, often seen in modern lifestyles, are a major social health concern. The aim of this study was to examine whether circadian rhythm disorders would influence angiogenesis and blood perfusion recovery in a mouse model of hind limb ischemia. Methods and Results A jet-lag model was established in C57BL/6J mice using a light-controlled isolation box. Control mice were kept at a light/dark 12:12 (12-hour light and 12-hour dark) condition. Concentrations of plasma vascular endothelial growth factor and circulating endothelial progenitor cells in control mice formed a circadian rhythm, which was diminished in the jet-lag model (P<0.05). The jet-lag condition deteriorated tissue capillary formation (P<0.001) and tissue blood perfusion recovery (P<0.01) in hind limb ischemia, which was associated with downregulation of vascular endothelial growth factor expression in local ischemic tissue and in the plasma. Although the expression of clock genes (ie, Clock, Bmal1, and Cry) in local tissues was upregulated after ischemic injury, the expression levels of cryptochrome (Cry) 1 and Cry2 were inhibited by the jet-lag condition. Next, Cry1 and Cry2 double-knockout mice were examined for blood perfusion recoveries and a reparative angiogenesis. Cry1 and Cry2 double-knockout mice revealed suppressed capillary density (P<0.001) and suppressed tissue blood perfusion recovery (P<0.05) in the hind limb ischemia model. Moreover, knockdown of CRY1/2 in human umbilical vein endothelial cells was accompanied by increased expression of WEE1 and decreased expression of HOXC5. This was associated with decreased proliferative capacity, migration ability, and tube formation ability of human umbilical vein endothelial cells, respectively, leading to impairment of angiogenesis. Conclusions Our data suggest that circadian rhythm disorder deteriorates reparative ischemia-induced angiogenesis and that maintenance of circadian rhythm plays an important role in angiogenesis.


Assuntos
Ritmo Circadiano , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Síndrome do Jet Lag/fisiopatologia , Neovascularização Fisiológica , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Criptocromos/genética , Criptocromos/metabolismo , Modelos Animais de Doenças , Células Progenitoras Endoteliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Isquemia/sangue , Isquemia/complicações , Isquemia/genética , Síndrome do Jet Lag/sangue , Síndrome do Jet Lag/complicações , Síndrome do Jet Lag/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Densidade Microvascular , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Fluxo Sanguíneo Regional , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/sangue
8.
Bioelectromagnetics ; 42(7): 593-602, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34289513

RESUMO

We previously found that a near-null magnetic field affected reproductive growth in Arabidopsis under white light. To test whether the effect of a near-null magnetic field on fruit growth of Arabidopsis is related to cryptochrome, we grew wild-type Arabidopsis and cryptochrome double mutant, cry1/cry2, in a near-null magnetic field under blue light. We found that fruit growth of wild-type Arabidopsis instead of the cry1/cry2 mutant was suppressed by the near-null magnetic field. Furthermore, gibberellin (GA) levels of GA4 , GA9 , GA34 , and GA51 in fruits of wild-type plants in the near-null magnetic fields were significantly lower than local geomagnetic field controls. However, in cry1/cry2 mutants, levels of the four detected GAs in fruits in the near-null magnetic fields did not differ significantly from controls. Expressions of GA20-oxidase (GA20ox) genes (GA20ox1 and GA20ox2) and GA3-oxidase (GA3ox) genes (GA3ox1 and GA3ox3) in fruits of wild-type plants rather than cry1/cry2 mutants were downregulated by the near-null magnetic field. In contrast, expressions of GA2-oxidase (GA2ox) genes and GA signaling genes were not affected by the near-null magnetic field. These results indicate that suppression of fruit growth by the near-null magnetic field is mediated by cryptochrome and that GAs are involved in the regulation of fruit growth by the near-null magnetic field. © 2021 Bioelectromagnetics Society.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Criptocromos/genética , Criptocromos/metabolismo , Frutas , Regulação da Expressão Gênica de Plantas , Luz , Campos Magnéticos
9.
Nat Commun ; 12(1): 3796, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145278

RESUMO

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.


Assuntos
Proteínas CLOCK/metabolismo , Relógios Circadianos/fisiologia , Criptocromos/metabolismo , Proteínas Circadianas Period/metabolismo , Análise de Célula Única/métodos , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Ritmo Circadiano/fisiologia , Criptocromos/genética , Técnicas de Introdução de Genes/métodos , Genes Reporter/genética , Células HCT116 , Humanos , Proteínas Circadianas Period/genética
10.
Photochem Photobiol Sci ; 20(6): 831-841, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34091863

RESUMO

Vibrio cholerae cryptochrome-1 (VcCRY-1) is a member of the cryptochrome DASH family. The flavoprotein appears to use blue light both for repair of cyclobutane pyrimidine dimers (CPDs) on DNA and signal transduction. Earlier, we found that it was almost impossible to oxidize the FADH· state upon binding to a CPD, and, in the absence of substrate, the rate of FADH· oxidation was much larger at high pH (Gindt et al. in Biochemistry 54:2802-2805, 2015). Here, we present the pH-dependence of the oxidation of FADH· by ferricyanide, which revealed a switch between slow and fast oxidation with a pKa ≈ 7.0. Stopped-flow mixing was used to measure the oxidation of FADH- to FADH· at pH 6.7 and 7.5. Substrate binding was required to slow down this oxidation such that it could be measured with stopped flow, but there was only a small effect of pH. In addition, resonance Raman measurements of FADH· in VcCRY-1 at pH 6.5 and 7.5 were performed to probe for structural changes near the FAD cofactor related to the observed changes in rate of FADH· oxidation. Only substrate binding seemed to induce a change near the FAD cofactor that may relate to the change in oxidation kinetics. The pH-effect on the FADH· oxidation rate, which is rate-limited by the proton acceptor, does not seem to be due to a protein structural change near the FAD cofactor. Instead, a conserved glutamate in CRY-DASH may control the deprotonation of FADH· and give rise to the pH-effect.


Assuntos
Criptocromos/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Ferricianetos/química , Concentração de Íons de Hidrogênio , Cinética , Oxirredução
11.
J Phys Chem Lett ; 12(23): 5558-5563, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34101477

RESUMO

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from Chlamydomonas reinhardtii in living bacterial cells, because the receptor degraded upon isolation. We demonstrate a stabilization of the flavin neutral radical as photoproduct and of the resulting ß-sheet reorganization by binding of cellular ATP. Comparison between light-induced structural responses of full-length pCRY and PHR reveals a downshift in frequency of the ß-sheet signal, implying an association of the CCT close to the only ß-sheet of the PHR in the dark. We provide a missing link in activation of plant cryptochromes after flavin photoreduction by indicating that ß-sheet reorganization causes the CCT release and restructuring.


Assuntos
Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Flavinas/química , Flavinas/metabolismo , Sítios de Ligação/fisiologia , Criptocromos/análise , Flavinas/análise , Conformação Proteica em Folha beta , Estrutura Secundária de Proteína , Espectrofotometria Infravermelho/métodos
12.
Nat Commun ; 12(1): 3593, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135337

RESUMO

Photoreceptors are conserved in green algae to land plants and regulate various developmental stages. In the ocean, blue light penetrates deeper than red light, and blue-light sensing is key to adapting to marine environments. Here, a search for blue-light photoreceptors in the marine metagenome uncover a chimeric gene composed of a phytochrome and a cryptochrome (Dualchrome1, DUC1) in a prasinophyte, Pycnococcus provasolii. DUC1 detects light within the orange/far-red and blue spectra, and acts as a dual photoreceptor. Analyses of its genome reveal the possible mechanisms of light adaptation. Genes for the light-harvesting complex (LHC) are duplicated and transcriptionally regulated under monochromatic orange/blue light, suggesting P. provasolii has acquired environmental adaptability to a wide range of light spectra and intensities.


Assuntos
Clorófitas/metabolismo , Oceanos e Mares , Fotorreceptores de Plantas/metabolismo , Fitoplâncton/metabolismo , Adaptação Fisiológica/genética , Núcleo Celular/metabolismo , Clorófitas/classificação , Clorófitas/genética , Criptocromos/genética , Criptocromos/metabolismo , Evolução Molecular , Luz , Metagenoma , Fotorreceptores de Plantas/genética , Filogenia , Fitocromo/genética , Fitocromo/metabolismo , Fitoplâncton/classificação , Fitoplâncton/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transcrição Genética/efeitos da radiação
13.
Sci Rep ; 11(1): 12683, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135416

RESUMO

Cryptochromes (CRY) have been proposed as putative magnetoreceptors in vertebrates. Localisation of CRY1 in the UV cones in the retinas of birds suggested that it could be the candidate magnetoreceptor. However, recent findings argue against this possibility. CRY1 is a type II cryptochrome, a subtype of cryptochromes that may not be inherently photosensitive, and it exhibits a clear circadian expression in the retinas of birds. Here, we reassessed the localisation and distribution of CRY1 in the retina of the zebra finch. Zebra finches have a light-dependent magnetic compass based on a radical-pair mechanism, similar to migratory birds. We found that CRY1 colocalised with the UV/V opsin (SWS1) in the outer segments of UV cones, but restricted to the tip of the segments. CRY1 was found in all UV cones across the entire retina, with the highest densities near the fovea. Pre-exposure of birds to different wavelengths of light did not result in any difference in CRY1 detection, suggesting that CRY1 did not undergo any detectable functional changes as result of light activation. Considering that CRY1 is likely not involved in magnetoreception, our findings open the possibility for an involvement in different, yet undetermined functions in the avian UV/V cones.


Assuntos
Proteínas Aviárias/metabolismo , Criptocromos/metabolismo , Tentilhões/metabolismo , Campos Magnéticos , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Luz
14.
Biosci Biotechnol Biochem ; 85(8): 1899-1909, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34124766

RESUMO

Light stimulates carotenoid production in an oleaginous yeast Rhodosporidium toruloides NBRC 10032 by promoting carotenoid biosynthesis genes. These genes undergo two-step transcriptional activation. The potential light regulator, Cryptochrome DASH (CRY1), has been suggested to contribute to this mechanism. In this study, based on KU70 (a component of nonhomologous end joining (NHEJ)) disrupting background, CRY1 disruptant was constructed to clarify CRY1 function. From analysis of CRY1 disruptant, it was suggested that CRY1 has the activation role of the carotenogenic gene expression. To obtain further insights into the light response, mutants varying carotenoid production were generated. Through analysis of mutants, the existence of the control two-step gene activation was proposed. In addition, our data analysis showed the strong possibility that R. toruloides NBRC 10032 is a homo-diploid strain.


Assuntos
Carotenoides/metabolismo , Luz , Rhodotorula/efeitos da radiação , Criptocromos/genética , Criptocromos/metabolismo , Marcação de Genes , Genes Fúngicos , Mutação , Polimorfismo de Nucleotídeo Único , Rhodotorula/genética , Rhodotorula/metabolismo
15.
J Neurosci ; 41(24): 5173-5189, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-33931552

RESUMO

We developed a method for single-cell resolution longitudinal bioluminescence imaging of PERIOD (PER) protein and TIMELESS (TIM) oscillations in cultured male adult Drosophila brains that captures circadian circuit-wide cycling under simulated day/night cycles. Light input analysis confirms that CRYPTOCHROME (CRY) is the primary circadian photoreceptor and mediates clock disruption by constant light (LL), and that eye light input is redundant to CRY; 3-h light phase delays (Friday) followed by 3-h light phase advances (Monday morning) simulate the common practice of staying up later at night on weekends, sleeping in later on weekend days then returning to standard schedule Monday morning [weekend light shift (WLS)]. PER and TIM oscillations are highly synchronous across all major circadian neuronal subgroups in unshifted light schedules for 11 d. In contrast, WLS significantly dampens PER oscillator synchrony and rhythmicity in most circadian neurons during and after exposure. Lateral ventral neuron (LNv) oscillations are the first to desynchronize in WLS and the last to resynchronize in WLS. Surprisingly, the dorsal neuron group-3 (DN3s) increase their within-group synchrony in response to WLS. In vivo, WLS induces transient defects in sleep stability, learning, and memory that temporally coincide with circuit desynchrony. Our findings suggest that WLS schedules disrupt circuit-wide circadian neuronal oscillator synchrony for much of the week, thus leading to observed behavioral defects in sleep, learning, and memory.


Assuntos
Encéfalo/fisiopatologia , Ritmo Circadiano/fisiologia , Criptocromos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas do Olho/metabolismo , Rede Nervosa/fisiopatologia , Proteínas Circadianas Period/metabolismo , Animais , Encéfalo/metabolismo , Drosophila , Aprendizagem/fisiologia , Masculino , Memória/fisiologia , Rede Nervosa/metabolismo , Sono/fisiologia
16.
Genes (Basel) ; 12(5)2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946956

RESUMO

Cryptochromes are flavin-containing blue light photoreceptors, present in most kingdoms, including archaea, bacteria, plants, animals and fungi. They are structurally similar to photolyases, a class of flavoproteins involved in light-dependent repair of UV-damaged DNA. Cryptochromes were first discovered in Arabidopsis thaliana in which they control many light-regulated physiological processes like seed germination, de-etiolation, photoperiodic control of the flowering time, cotyledon opening and expansion, anthocyanin accumulation, chloroplast development and root growth. They also regulate the entrainment of plant circadian clock to the phase of light-dark daily cycles. Here, we review the molecular mechanisms by which plant cryptochromes control the synchronisation of the clock with the environmental light. Furthermore, we summarise the circadian clock-mediated changes in cell cycle regulation and chromatin organisation and, finally, we discuss a putative role for plant cryptochromes in the epigenetic regulation of genes.


Assuntos
Relógios Circadianos , Criptocromos/metabolismo , Proteínas de Plantas/metabolismo , Criptocromos/genética , Epigênese Genética , Proteínas de Plantas/genética , Plantas/genética , Plantas/metabolismo
17.
Plant Cell Physiol ; 62(6): 1001-1011, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34043001

RESUMO

Eggplant is rich in anthocyanins, which are thought to be highly beneficial for human health. It has been reported that blue light inhibitors of cryptochromes (BICs) act as negative regulators in light signal transduction, but little is known about their role in anthocyanin biosynthesis. In this study, yeast one-hybrid analysis showed that SmBICs could bind to the promoter of SmCHS, indicating that they could directly participate in eggplant anthocyanin biosynthesis. In SmBICs-silenced eggplants, more anthocyanins were accumulated, while SmBIC1-overexpression (OE) and SmBIC2-OE Arabidopsis and eggplants synthesized less anthocyanin. Quantitative real-time polymerase chain reaction also revealed that the anthocyanin structural genes, which were downregulated in SmBIC1-OE and SmBIC2-OE lines, were upregulated in SmBICs-silenced eggplants. In addition, transcriptome analysis further confirmed that differentially expressed genes of SmBICs-OE plants were enriched mainly in the pathways related to anthocyanin biosynthesis and the key transcription factors and structural genes for anthocyanin biosynthesis, such as SmMYB1, SmTT8, SmHY5, SmCHS, SmCHI, SmDFR and SmANS, were suppressed significantly. Finally, bimolecular fluorescence complementation and blue-light-dependent degradation assay suggested that SmBICs interacted with photo-excited SmCRY2 to inhibit its photoreaction, thereby inhibiting the expression of genes related to anthocyanin biosynthesis and reducing anthocyanin accumulation. Collectively, our study suggests that SmBICs repress anthocyanin biosynthesis by inhibiting photoactivation of SmCRY2. This study provides a new working model for anthocyanin biosynthesis in eggplant.


Assuntos
Antocianinas/biossíntese , Proteínas de Plantas/metabolismo , Solanum melongena/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Criptocromos/genética , Criptocromos/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Transdução de Sinal Luminoso , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Solanum melongena/fisiologia , Ativação Transcricional
18.
Nat Commun ; 12(1): 2155, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846325

RESUMO

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1/SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1/SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Criptocromos/metabolismo , Fotorreceptores de Plantas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Criptocromos/química , Criptocromos/genética , Células HEK293 , Humanos , Luz , Modelos Biológicos , Mutação/genética , Fosforilação/efeitos da radiação , Poliubiquitina/metabolismo , Ligação Proteica/efeitos da radiação , Proteólise/efeitos da radiação , Plântula/efeitos da radiação , Ubiquitinação/efeitos da radiação
19.
Mol Med ; 27(1): 43, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33902432

RESUMO

BACKGROUND: Osteoporosis seriously disturbs the life of people. Meanwhile, inhibition or weakening of osteogenic differentiation is one of the important factors in the pathogenesis of osteoporosis. It was reported that miR-27a-3p reduced the symptoms of osteoporosis. However, the mechanism by which miR-27a-3p in osteogenic differentiation remains largely unknown. METHODS: To induce the osteogenic differentiation in MC3T3-E1 cells, cells were treated with osteogenic induction medium (OIM). RT-qPCR was used to evaluate the mRNA expression of miR-27a-3p and CRY2 in cells. The protein levels of CRY2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and the phosphorylation level of extracellular regulated protein kinases (ERK) 1/2 in MC3T3-E1 cells were evaluated by western blotting. Meanwhile, calcium nodules and ALP activity were tested by alizarin red staining and ALP kit, respectively. Luciferase reporter gene assay was used to analyze the correlation between CRY2 and miR-27a-3p. RESULTS: The expression of miR-27a-3p and the phosphorylation level of ERK1/2 were increased by OIM in MC3T3-E1 cells, while CRY2 expression was decreased. In addition, OIM-induced increase of calcified nodules, ALP content and osteogenesis-related protein expression was significantly reversed by downregulation of miR-27a-3p and overexpression of CRY2. In addition, miR-27a-3p directly targeted CRY2 and negatively regulated CRY2. Meanwhile, the inhibitory effect of miR-27a-3p inhibitor on osteogenic differentiation was reversed by knockdown of CRY2 or using honokiol (ERK1/2 signal activator). Furthermore, miR-27a-3p significantly inhibited the apoptosis of MC3T3-E1 cells treated by OIM. Taken together, miR-27a-3p/CRY2/ERK axis plays an important role in osteoblast differentiation. CONCLUSIONS: MiR-27a-3p promoted osteoblast differentiation via mediation of CRY2/ERK1/2 axis. Thereby, miR-27a-3p might serve as a new target for the treatment of osteoporosis.


Assuntos
MicroRNAs , Osteoblastos/citologia , Osteogênese/genética , Animais , Apoptose/genética , Autofagia/genética , Diferenciação Celular/genética , Linhagem Celular , Criptocromos/genética , Criptocromos/metabolismo , Regulação para Baixo , Sistema de Sinalização das MAP Quinases , Camundongos
20.
Plant Cell Environ ; 44(6): 1802-1815, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33665849

RESUMO

Cryptochromes photoreceptors, CRY1 and CRY2 in Arabidopsis, mediate blue light responses in plants and metazoa. The signalling interactions underlying photomorphogenesis of cryptochromes action have been extensively studied in experiment, expecting a systematical analysis of the dynamic mechanisms of photosensory signalling network from a global view. In this study, we developed a signalling network model to quantitatively investigate the different response modes and cooperation modulations on photomorphogenesis for CRY1 and CRY2 under blue light. The model shows that the different modes of time-dependent and fluence-rate-dependent phosphorylations for CRY1 and CRY2 are originated from their different phosphorylation rates and degradation rates. Our study indicates that, due to the strong association between blue-light inhibitor of cryptochromes (BIC) and CRY2, BIC negatively modulates CRY2 phosphorylation, which was confirmed by our experiment. The experiment also validated the model prediction that the time-dependent BIC-CRY1 and the fluence-rate-dependent BIC-CRY2 are both bell-shaped under blue light. Importantly, the model proposes that the COP1-SPA abundance can strongly inhibit the phosphorylation response of CRY2, resulting in the positive regulation of CRY2 phosphorylation by CRY1 through COP1-SPA. The model also predicts that the CRY1-HY5 axis, rather than CRY2-HY5 pathway, plays a dominant role in blue-light-dependent photomorphogenesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Criptocromos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Criptocromos/genética , Células HEK293 , Humanos , Luz , Morfogênese , Mutação , Fosforilação , Plantas Geneticamente Modificadas , Fatores de Tempo , Ubiquitina-Proteína Ligases/metabolismo
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