RESUMEN
Cryptochromes (CRYs) are essential components of the circadian clock, playing a pivotal role as transcriptional repressors. Despite their significance, the precise mechanisms underlying CRYs' involvement in the circadian clock remain incompletely understood. In this study, we identified a rare CRY2 variant, p.Ser420Phe, from the 1000 Genomes Project and Ensembl database that is located in the functionally important coiled-coil-like helix (CC-helix) region. Functional characterization of this variant at the cellular level revealed that p.Ser420Phe CRY2 had reduced repression activity on CLOCK:BMAL1-driven transcription due to its reduced affinity to the core clock protein PER2 and defective translocation into the nucleus. Intriguingly, the CRY2 variant exhibited an unexpected resistance to degradation via the canonical proteasomal pathway, primarily due to the loss of interactions with E3 ligases (FBXL3 and FBXL21), which suggests Ser-420 of CRY2 is required for the interaction with E3 ligases. Further studies revealed that wild-type and CRY2 variants are degraded by the lysosomal-mediated degradation pathway, a mechanism not previously associated with CRY2. Surprisingly, our complementation study with Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cells indicated that the CRY2 variant caused a 7 h shorter circadian period length in contrast to the observed prolonged period length in CRY2-/- cell lines. In summary, this study reveals a hitherto unknown degradation pathway for CRY2, shedding new light on the regulation of circadian rhythm period length.
Asunto(s)
Sustitución de Aminoácidos , Relojes Circadianos , Criptocromos , Animales , Humanos , Ratones , Relojes Circadianos/fisiología , Ritmo Circadiano/fisiología , Proteínas CLOCK/metabolismo , Criptocromos/genética , Criptocromos/metabolismo , Fibroblastos/metabolismo , Lisosomas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea CelularRESUMEN
Cryptochromes (CRYs), transcriptional repressors of the circadian clock in mammals, inhibit cAMP production when glucagon activates G-protein coupled receptors. Therefore, molecules that modulate CRYs have the potential to regulate gluconeogenesis. In this study, we discovered a new molecule called TW68 that interacts with the primary pockets of mammalian CRY1/2, leading to reduced ubiquitination levels and increased stability. In cell-based circadian rhythm assays using U2OS Bmal1-dLuc cells, TW68 extended the period length of the circadian rhythm. Additionally, TW68 decreased the transcriptional levels of two genes, Phosphoenolpyruvate carboxykinase 1 (PCK1) and Glucose-6-phosphatase (G6PC), which play crucial roles in glucose biosynthesis during glucagon-induced gluconeogenesis in HepG2 cells. Oral administration of TW68 in mice showed good tolerance, a good pharmacokinetic profile, and remarkable bioavailability. Finally, when administered to fasting diabetic animals from ob/ob and HFD-fed obese mice, TW68 reduced blood glucose levels by enhancing CRY stabilization and subsequently decreasing the transcriptional levels of Pck1 and G6pc. These findings collectively demonstrate the antidiabetic efficacy of TW68 in vivo, suggesting its therapeutic potential for controlling fasting glucose levels in the treatment of type 2 diabetes mellitus.
Asunto(s)
Relojes Circadianos , Diabetes Mellitus Tipo 2 , Animales , Ratones , Criptocromos/genética , Glucemia , Ratones Obesos , Glucagón , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Ritmo Circadiano/fisiología , Mamíferos , AyunoRESUMEN
Cryptochromes (CRYs) are essential components of the molecular clock that generates circadian rhythm. They inhibit BMAL1/CLOCK-driven transcription at the molecular level. There are two CRYs that have differential functions in the circadian clock in mammals. It is not precisely known how they achieve such differential functions. In this study, we performed molecular dynamic simulations on eight CRY mutants that have been experimentally shown to exhibit reduced repressor activities. Our results revealed that mutations in CRY1 affect the dynamic behavior of the serine loop and the availability of the secondary pocket, but not in CRY2. Further analysis of these CRY1 mutants indicated that the differential flexibility of the serine loop leads to changes in the volume of the secondary pocket. We also investigated the weak interactions between the amino acids in the serine loop and those in close proximity. Our findings highlighted the crucial roles of S44 and S45 in the dynamic behavior of the serine loop, specifically through their interactions with E382 in CRY1. Considering the clinical implications of altered CRY1 function, our study opens up new possibilities for the development of drugs that target the allosteric regulation of CRY1.Communicated by Ramaswamy H. Sarma.
RESUMEN
The circadian timing system controls absorption, distribution, metabolism, and elimination processes of drug pharmacokinetics over a 24-h period. Exposure of target tissues to the active form of the drug and cytotoxicity display variations depending on the chronopharmacokinetics. For anticancer drugs with narrow therapeutic ranges and dose-limiting side effects, it is particularly important to know the temporal changes in pharmacokinetics. A previous study indicated that pharmacokinetic profile of capecitabine was different depending on dosing time in rat. However, it is not known how such difference is attributed with respect to diurnal rhythm. Therefore, in this study, we evaluated capecitabine-metabolizing enzymes in a diurnal rhythm-dependent manner. To this end, C57BL/6J male mice were orally treated with 500 mg/kg capecitabine at ZT1, ZT7, ZT13, or ZT19. We then determined pharmacokinetics of capecitabine and its metabolites, 5'-deoxy-5-fluorocytidine (5'DFCR), 5'-deoxy-5-fluorouridine (5'DFUR), 5-fluorouracil (5-FU), in plasma and liver. Results revealed that plasma Cmax and AUC0-6h (area under the plasma concentration-time curve from 0 to 6 h) values of capecitabine, 5'DFUR, and 5-FU were higher during the rest phase (ZT1 and ZT7) than the activity phase (ZT13 and ZT19) (p < 0.05). Similarly, Cmax and AUC0-6h values of 5'DFUR and 5-FU in liver were higher during the rest phase than activity phase (p < 0.05), while there was no significant difference in liver concentrations of capecitabine and 5'DFCR. We determined the level of the enzymes responsible for the conversion of capecitabine and its metabolites at each ZT. Results indicated the levels of carboxylesterase 1 and 2, cytidine deaminase, uridine phosphorylase 2, and dihydropyrimidine dehydrogenase (p < 0.05) are being rhythmically regulated and, in turn, attributed different pharmacokinetics profiles of capecitabine and its metabolism. This study highlights the importance of capecitabine administration time to increase the efficacy with minimum adverse effects.
Asunto(s)
Antimetabolitos Antineoplásicos , Ritmo Circadiano , Masculino , Ratones , Ratas , Animales , Capecitabina/farmacocinética , Antimetabolitos Antineoplásicos/farmacocinética , Antimetabolitos Antineoplásicos/uso terapéutico , Ratones Endogámicos C57BL , Fluorouracilo/metabolismo , Fluorouracilo/uso terapéuticoRESUMEN
Cryptochromes are negative transcriptional regulators of the circadian clock in mammals. It is not clear how reducing the level of endogenous CRY1 in mammals will affect circadian rhythm and the relation of such a decrease with apoptosis. Here, we discovered a molecule (M47) that destabilizes Cryptochrome 1 (CRY1) both in vitro and in vivo. The M47 selectively enhanced the degradation rate of CRY1 by increasing its ubiquitination and resulted in increasing the circadian period length of U2OS Bmal1-dLuc cells. In addition, subcellular fractionation studies from mice liver indicated that M47 increased degradation of the CRY1 in the nucleus. Furthermore, M47-mediated CRY1 reduction enhanced oxaliplatin-induced apoptosis in Ras-transformed p53 null fibroblast cells. Systemic repetitive administration of M47 increased the median lifespan of p53-/- mice by ~25%. Collectively our data suggest that M47 is a promising molecule to treat forms of cancer depending on the p53 mutation.
Asunto(s)
Relojes Circadianos , Criptocromos , Animales , Ratones , Relojes Circadianos/genética , Ritmo Circadiano/genética , Criptocromos/genética , Criptocromos/metabolismo , Longevidad , Mamíferos/metabolismo , Ratones Noqueados , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Human clock-gene variations contribute to the phenotypic differences observed in various behavioral and physiological processes, such as diurnal preference, sleep, metabolism, mood regulation, addiction, and fertility. However, little is known about the possible effects of identified variations at the molecular level. In this study, we performed a functional characterization at the cellular level of rare cryptochrome 2 (CRY2) missense variations that were identified from the Ensembl database. Our structural studies revealed that three variations (p.Pro123Leu, p.Asp406His, and p.Ser410Ile) are located at the rim of the secondary pocket of CRY2. We show that these variants were unable to repress CLOCK (circadian locomotor output cycles kaput)/BMAL1 (brain and muscle ARNT-like-1)-driven transcription in a cell-based reporter assay and had reduced affinity to CLOCK-BMAL1. Furthermore, our biochemical studies indicated that the variants were less stable than the WT CRY2, which could be rescued in the presence of period 2 (PER2), another core clock protein. Finally, we found that these variants were unable to properly localize to the nucleus and thereby were unable to rescue the circadian rhythm in a Cry1-/-Cry2-/- double KO mouse embryonic fibroblast cell line. Collectively, our data suggest that the rim of the secondary pocket of CRY2 plays a significant role in its nuclear localization independently of PER2 and in the intact circadian rhythm at the cellular level.
Asunto(s)
Factores de Transcripción ARNTL , Proteínas CLOCK , Ritmo Circadiano , Criptocromos , Factores de Transcripción ARNTL/metabolismo , Animales , Proteínas CLOCK/metabolismo , Ritmo Circadiano/fisiología , Criptocromos/química , Criptocromos/genética , Criptocromos/metabolismo , Fibroblastos , Humanos , Ratones , Dominios Proteicos , Estabilidad ProteicaRESUMEN
Heterogeneity in symptoms associated with COVID-19 in infected patients remains unclear. ACE2 and TMPRSS2 gene variants are considered possible risk factors for COVID-19. In this study, a retrospective comparative genome analysis of the ACE2 and TMPRSS2 variants from 946 whole-exome sequencing data was conducted. Allele frequencies of all variants were calculated and filtered to remove variants with allele frequencies lower than 0.003 and to prioritize functional coding variants. The majority of detected variants were intronic, only two ACE2 and three TMPRSS2 nonsynonymous variants were detected in the analyzed cohort. The main ACE2 variants that putatively have a protective or susceptibility effect on SARS-CoV-2 have not yet been determined in the Turkish population. The Turkish genetic makeup likely lacks any ACE2 variant that increases susceptibility to SARS-CoV-2 infection. TMPRSS2 rs75603675 and rs12329760 variants that were previously defined as common variants that have different allele frequencies among populations and may have a role in SARS-CoV-2 attachment to host cells were determined in the population. Overall, these data will contribute to the formation of a national variation database and may also contribute to further studies of ACE2 and TMPRSS2 in the Turkish population and differences in SARS-CoV-2 infection among other populations.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/genética , COVID-19/epidemiología , COVID-19/genética , Humanos , Peptidil-Dipeptidasa A/genética , Estudios Retrospectivos , Factores de Riesgo , SARS-CoV-2/genética , Serina Endopeptidasas/genética , Secuenciación del ExomaRESUMEN
Circadian rhythms are part of the body's clock, which regulates several physiological and biochemical variables according to the 24-h cycle. Ample evidence indicated disturbance of the circadian clock leads to an increased susceptibility to several diseases. Therefore, a great effort has been made to find small molecules that regulate circadian rhythm by high-throughput methods. Having crystal structures of core clock proteins, makes them amenable to structure-based drug design studies. Here, we describe virtual screening methods that can be utilized for the identification of small molecules regulating the activity of core clock protein Cryptochrome 1.
Asunto(s)
Proteínas CLOCK , Relojes Circadianos , Proteínas CLOCK/metabolismo , Ritmo Circadiano/fisiología , Simulación del Acoplamiento MolecularRESUMEN
Circadian rhythms are a series of endogenous autonomous 24-h oscillations generated by the circadian clock. At the molecular level, the circadian clock is based on a transcription-translation feedback loop, in which BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME (CRY) and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. There are three PER proteins, of which PER2 shows the strongest oscillation at both stability and cellular localization level. Protein-protein interactions (PPIs) or interactome of the circadian clock proteins have been investigated using classical methods such as two-dimensional gel electrophoresis, immunoprecipitation-coupled mass spectrometry, and yeast-two hybrid assay where the dynamic and weak interactions are difficult to catch. To identify the interactome of PER2 we have adopted proximity-dependent labeling with biotin and mass spectrometry-based identification of labeled proteins (BioID). In addition to known interactions with such as CRY1 and CRY2, we have identified several new PPIs for PER2 and confirmed some of them using co-immunoprecipitation technique. This study characterizes the PER2 protein interactions in depth, and it also implies that using a fast BioID method with miniTurbo or TurboID coupled to other major circadian clock proteins might uncover other interactors in the clock that have yet to be discovered.
Asunto(s)
Relojes Circadianos , Proteínas Circadianas Period , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relojes Circadianos/genética , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Proteoma/metabolismoRESUMEN
Birt-Hogg-Dube syndrome (BHDS) is a rare disorder characterized by the triad of cutaneous lesions, renal tumors, lung cysts and inactivation of the gene folliculin (FLCN). Here, we present three female patients diagnosed with BHDS. First case a 55-year-old female had flesh moles histopathology compatible with angiofibroma, multiple cysts in the lung and kidneys, FLCN gene mutations ('c.1285dupC [p.His429Profs*]' 11th exon and 'c.653G>A [p.Arg258His]' 7th exon). The second case a 76-year-old female had trichodiscoma on her skin, multiple cysts in the lung, spontaneous pneumothorax, FLCN gene mutation 'c.1285dupC (p.His429Profs*27) 11th exon' and, her son had renal carcinoma history under 50 years of age. Our third case, also the daughter of case 2, had dermal papules histopathology compatible with trichodiscoma, spontaneous pneumothorax, FLCN gene mutation 'c.1285dupC (p.His429Profs*27) 11th exon' and, parotid oncocytoma. Through our cases, we document the first case of two mutations ('c.1285dupC [p.His429Profs*]' 11th exon and 'c.653G>A [p.Arg258His]' 7th exon) in the same FLCN gene and the 11th known case of parotid oncocytoma associated with BHDS in the light of the literature.
RESUMEN
Ornithine transcarbamylase deficiency (OTCD) is the most common X-linked hereditary disorder of urea cycle disorders that is caused by neonatal hyperammonemia. OTC gene sequence variations are common causes of OTCD. The current study presents a 28-month-old baby girl proband with phenotypical characteristics of OTCD such as irritability, somnolence, intermittent vomiting, and high levels of serum ammonium. Whole-exome sequencing revealed a de novo c.275G>A p.(Arg92Gln) variant within the OTC gene. In silico analysis revealed a possible differential affinity between wild-type and mutant OTCase, while Arg92Gln decreases the binding ability of OTCase to the substrate, which can disrupt the urea cycle and explains the molecular pathogenicity of clinical hyperammonemia. In light of the fact that the genotype and phenotype correlation of OTCD is still uncertain, the present in silico analysis outcome can enhance our knowledge on this complicated, rare, and severe genetic disorder.
Asunto(s)
Hiperamonemia , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa , Humanos , Hiperamonemia/complicaciones , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/complicaciones , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/genética , Secuenciación del ExomaRESUMEN
Despite COVID-19 turned into a pandemic, no approved drug for the treatment or globally available vaccine is out yet. In such a global emergency, drug repurposing approach that bypasses a costly and long-time demanding drug discovery process is an effective way in search of finding drugs for the COVID-19 treatment. Recent studies showed that SARS-CoV-2 uses neuropilin-1 (NRP1) for host entry. Here we took advantage of structural information of the NRP1 in complex with C-terminal of spike (S) protein of SARS-CoV-2 to identify drugs that may inhibit NRP1 and S protein interaction. U.S. Food and Drug Administration (FDA) approved drugs were screened using docking simulations. Among top drugs, well-tolerated drugs were selected for further analysis. Molecular dynamics (MD) simulations of drugs-NRP1 complexes were run for 100 ns to assess the persistency of binding. MM/GBSA calculations from MD simulations showed that eltrombopag, glimepiride, sitagliptin, dutasteride, and ergotamine stably and strongly bind to NRP1. In silico Alanine scanning analysis revealed that Tyr297, Trp301, and Tyr353 amino acids of NRP1 are critical for drug binding. Validating the effect of drugs analyzed in this paper by experimental studies and clinical trials will expedite the drug discovery process for COVID-19.
RESUMEN
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.
Asunto(s)
Proteínas CLOCK/metabolismo , Ritmo Circadiano/fisiología , Criptocromos/metabolismo , Animales , Bases de Datos de Proteínas , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Next-generation sequencing technology and advanced sequence analysis techniques are markedly speeding up the identification of gene variants causing rare genetic diseases. Pseudoachondroplasia (PSACH, MIM 177170) is a rare disease inherited in an autosomal dominant manner. It is known that variations in the cartilage oligomeric matrix protein (COMP) gene are associated with the disease. Here, we report a 39-month-old boy with short stature. He gave visible growth and development delayed phenotype after 12 months. Further genetic resequencing analysis was carried out to identified the disease-causing variant. Furthermore, computational approaches were used to characterize the effect of the variant. In this study, we identify and report a novel variation in the COMP gene, c.1420_1422del (p.Asn47del), causing a spontaneous form of PSACH in our patient. Our in silico model indicated that any mutational changes in this region are very susceptible to PASCH phenotype. Overall, this study is the first PSACH case in the Turkish Cypriot population. Moreover, this finding contributes to the concept that the genotype-phenotype correlation in COMP is still unknown and also improves our understanding of this complex disorder.
Asunto(s)
Acondroplasia/genética , Secuencia de Aminoácidos , Proteína de la Matriz Oligomérica del Cartílago/genética , Eliminación de Secuencia , Preescolar , Humanos , MasculinoRESUMEN
Despite strict measures taken by many countries, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be an issue of global concern. Currently, there are no clinically proven pharmacotherapies for coronavirus disease 2019, despite promising initial results obtained from drugs such as azithromycin and hydroxychloroquine. Therefore, the repurposing of clinically approved drugs for use against SARS-CoV-2 has become a viable strategy. Here, we searched for drugs that target SARS-CoV-2 3C-like protease (3CLpro) and viral RNA-dependent RNA polymerase (RdRp) by in silico screening of the U.S. Food and Drug Administration approved drug library. Well-tolerated and widely used drugs were selected for molecular dynamics (MD) simulations to evaluate drug-protein interactions and their persistence under physiological conditions. Tetracycline, dihydroergotamine, ergotamine, dutasteride, nelfinavir, and paliperidone formed stable interactions with 3CLpro based on MD simulation results. Similar analysis with RdRp showed that eltrombopag, tipranavir, ergotamine, and conivaptan bound to the enzyme with high binding free energies. Docking results suggest that ergotamine, dihydroergotamine, bromocriptine, dutasteride, conivaptan, paliperidone, and tipranavir can bind to both enzymes with high affinity. As these drugs are well tolerated, cost-effective, and widely used, our study suggests that they could potentially to be used in clinical trials for the treatment of SARS-CoV-2-infected patients.Communicated by Ramaswamy H. Sarma.
Asunto(s)
COVID-19 , Preparaciones Farmacéuticas , Antivirales , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Péptido Hidrolasas , Inhibidores de Proteasas , ARN Polimerasa Dependiente del ARN , SARS-CoV-2RESUMEN
We have previously reported that the deletion of BMAL1 gene has opposite effects in respect to its contribution to the pathways that are effective in the multistage carcinogenesis process. BMAL1 deletion sensitized nearly normal breast epithelial (MCF10A) and invasive breast cancer cells (MDA-MB-231) to cisplatin- and doxorubicin-induced apoptosis, while this deletion also aggravated the invasive potential of MDA-MB-231 cells. However, the mechanistic relationship of the seemingly opposite contribution of BMAL1 deletion to carcinogenesis process is not known at genome-wide level. In this study, an RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways after treating BMAL1 knockout (KO) or wild-type (WT) MDA-MB-231 cells with cisplatin and doxorubicin to initiate apoptosis. Gene set enrichment analysis with the DEGs demonstrated that enrichment in multiple genes/pathways contributes to sensitization to cisplatin- or doxorubicin-induced apoptosis in BMAL1-dependent manner. Additionally, our DEG analysis suggested that non-coding transcript RNA (such as lncRNA and processed pseudogenes) may have role in cisplatin- or doxorubicin-induced apoptosis. Protein-protein interaction network obtained from common DEGs in cisplatin and doxorubicin treatments revealed that GSK3ß, NACC1, and EGFR are the principal genes regulating the response of the KO cells. Moreover, the analysis of DEGs among untreated BMAL1 KO and WT cells revealed that epithelial-mesenchymal transition genes are up-regulated in KO cells. As a negative control, we have also analyzed the DEGs following treatment with an endoplasmic reticulum (ER) stress-inducing agent, tunicamycin, which was affected by BMAL1 deletion minimally. Collectively, the present study suggests that BMAL1 regulates many genes/pathways of which the alteration in BMAL1 KO cells may shed light on pleotropic phenotype observed.
Asunto(s)
Factores de Transcripción ARNTL/genética , Carcinogénesis/genética , Transcriptoma , Factores de Transcripción ARNTL/metabolismo , Apoptosis , Carcinogénesis/metabolismo , Línea Celular Tumoral , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , HumanosRESUMEN
Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box-driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg-293 of CRY1, identified as a rare CRY1 SNP in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/CLOCK in the absence of PER2 compared with CRY1. Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg-293 and the serine loop by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm.
Asunto(s)
Proteínas CLOCK , Ritmo Circadiano , Criptocromos , Simulación de Dinámica Molecular , Factores de Transcripción ARNTL/química , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Regulación Alostérica , Sustitución de Aminoácidos , Animales , Arginina/química , Arginina/genética , Arginina/metabolismo , Proteínas CLOCK/química , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Criptocromos/química , Criptocromos/genética , Criptocromos/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Mutación Missense , Proteínas Circadianas Period/química , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Polimorfismo de Nucleótido Simple , Unión Proteica , Estructura Secundaria de Proteína , Transcripción GenéticaRESUMEN
Neonatal progeroid syndrome or Wiedemann-Rautenstrauch syndrome (WRS; MIM 264090) is a rare genetic disorder that has clinical symptoms including premature aging, lipodystrophy, and variable mental impairment. Until recently genetic background of the disease was unclear. However, recent studies have indicated that WRS patients have compound heterozygote variations in the POLR3A (RNA polymerase III subunit 3A; MIM 614258) gene that might be responsible for the disease phenotype. In this study we report a WRS patient that has compound heterozygote variations in the POLR3A gene. One of the reported variations in our patient, c.3568C>T, p.(Gln1190Ter), is a novel variation that was not reported before. The other variant, c.3337-11T>C, was previously shown in WRS patients in trans with other variations.
Asunto(s)
Retardo del Crecimiento Fetal/genética , Progeria/genética , ARN Polimerasa III/genética , Alelos , Niño , Femenino , Retardo del Crecimiento Fetal/patología , Humanos , Simulación de Dinámica Molecular , Mutación , Progeria/patología , ARN Polimerasa III/químicaRESUMEN
Attention deficit/hyperactivity disorder (ADHD) is a common and heritable phenotype frequently accompanied by insomnia, anxiety, and depression. Here, using a reverse phenotyping approach, we report heterozygous coding variations in the core circadian clock gene cryptochrome 1 in 15 unrelated multigenerational families with combined ADHD and insomnia. The variants led to functional alterations in the circadian molecular rhythms, providing a mechanistic link to the behavioral symptoms. One variant, CRY1Δ11 c.1657+3A>C, is present in approximately 1% of Europeans, therefore standing out as a diagnostic and therapeutic marker. We showed by exome sequencing in an independent cohort of patients with combined ADHD and insomnia that 8 of 62 patients and 0 of 369 controls carried CRY1Δ11. Also, we identified a variant, CRY1Δ6 c.825+1G>A, that shows reduced affinity for BMAL1/CLOCK and causes an arrhythmic phenotype. Genotype-phenotype correlation analysis revealed that this variant segregated with ADHD and delayed sleep phase disorder (DSPD) in the affected family. Finally, we found in a phenome-wide association study involving 9438 unrelated adult Europeans that CRY1Δ11 was associated with major depressive disorder, insomnia, and anxiety. These results defined a distinctive group of circadian psychiatric phenotypes that we propose to designate as "circiatric" disorders.
Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/genética , Criptocromos/genética , Mutación , Trastornos del Sueño del Ritmo Circadiano/genética , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Adulto , Trastorno por Déficit de Atención con Hiperactividad/metabolismo , Trastorno por Déficit de Atención con Hiperactividad/patología , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Criptocromos/metabolismo , Femenino , Estudios de Asociación Genética , Células HEK293 , Humanos , Masculino , Trastornos del Sueño del Ritmo Circadiano/metabolismoRESUMEN
Proper function of many physiological processes requires a robust circadian clock. Disruptions of the circadian clock can result in metabolic diseases, mood disorders, and accelerated aging. Therefore, identifying small molecules that specifically modulate regulatory core clock proteins may potentially enable better management of these disorders. In this study, we applied a structure-based molecular-docking approach to find small molecules that specifically bind to the core circadian regulator, the transcription factor circadian locomotor output cycles kaput (CLOCK). We identified 100 candidate molecules by virtual screening of â¼2 million small molecules for those predicted to bind closely to the interface in CLOCK that interacts with its transcriptional co-regulator, Brain and muscle Arnt-like protein-1 (BMAL1). Using a mammalian two-hybrid system, real-time monitoring of circadian rhythm in U2OS cells, and various biochemical assays, we tested these compounds experimentally and found one, named CLK8, that specifically bound to and interfered with CLOCK activity. We show that CLK8 disrupts the interaction between CLOCK and BMAL1 and interferes with nuclear translocation of CLOCK both in vivo and in vitro Results from further experiments indicated that CLK8 enhances the amplitude of the cellular circadian rhythm by stabilizing the negative arm of the transcription/translation feedback loop without affecting period length. Our results reveal CLK8 as a tool for further studies of CLOCK's role in circadian rhythm amplitude regulation and as a potential candidate for therapeutic development to manage disorders associated with dampened circadian rhythms.