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While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2-27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal transition (EMT) and whose formation depends on the epithelial splicing regulator ESRP1. CircDOCK1(2-27) synthesis in epithelial cells represses cell motility both by diverting transcripts from DOCK1 mRNA production to circRNA formation and by direct inhibition of migration by the circRNA. HITS-CLIP analysis and CRISPR-mediated deletions indicate ESRP1 controls circDOCK1(2-27) biosynthesis by binding a GGU-containing repeat region in intron 1 and detaining its splicing until Pol II completes its 157 kb journey to exon 27. Proximity-dependent biotinylation (BioID) assay suggests ESRP1 may modify the RNP landscape of intron 1 in a way that disfavours communication of exon 1 with exon 2, rather than physically bridging exon 2 to exon 27. The X-ray crystal structure of RNA-bound ESRP1 qRRM2 domain reveals it binds to GGU motifs, with the guanines embedded in clamp-like aromatic pockets in the protein.
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Processamento Alternativo , RNA Circular , Proteínas de Ligação a RNA , Proteínas rac de Ligação ao GTP , RNA/genética , RNA/metabolismo , Splicing de RNA , RNA Circular/genética , Humanos , Linhagem Celular Tumoral , Proteínas de Ligação a RNA/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
TDP-43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP-43, are one of the common causes of familial ALS. In this study, we investigate TDP-43 protein behavior in induced pluripotent stem cell (iPSC)-derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP-43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP-43, C-terminal fragments, and phospho-TDP-43. By generating iPSC lines with allele-specific tagging of TDP-43, we find that mutant TDP-43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP-43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP-43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP-43 pathology.
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Esclerose Lateral Amiotrófica/metabolismo , Transporte Axonal , Proteínas de Ligação a DNA/genética , Desacetilase 6 de Histona/metabolismo , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/genética , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Mutação de Sentido IncorretoRESUMO
BACKGROUND: PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS: We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.
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Canal de Liberação de Cálcio do Receptor de Rianodina , Serina , Camundongos , Animais , Humanos , Isoproterenol/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Serina/metabolismo , Serina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Isoquinolinas/farmacologia , Sulfonamidas/farmacologia , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
A Pd-catalyzed dual C-H carbonylation of commercially available diarylamines using Co2(CO)8 as a safe CO source has been developed. This methodology provides a facile approach for the synthesis of diversified acridones in moderate to good yields. The protocol features good functional group compatibility, operational safety, easy scale-up, and versatile transformations.
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There is unclear evidence available on the associations between multiple metals and fasting blood glucose (FBG) in children, and whether they could be beneficial from physical activity. We included 283 children aged 4-12 years from two panel studies with 4-consecutive morning urinary 13 essential metals and 10 non-essential metals repeated across 3 seasons. We employed multiple informant model, linear mixed-effect model, and quantile g-computation to evaluate associations of single metal and their mixture with FBG and interactions with extra-school activity. The results showed that positive relations of multiple essential metals (aluminum, chromium, copper, iron, molybdenum (Mo), nickel, selenium (Se), strontium, zinc) and non-essential metals (arsenic (As), cadmium (Cd), rubidium, titanium (Ti), thallium) with FBG were the strongest at lag 0 (the health examination day), especially in overweight & obesity children (FDR <0.05). The strongest effect presented 1-fold increment in As was related to FBG increased 1.66% (95%CI: 0.84%, 2.48%) in overweight & obesity children. Notably, modification of extra-school activity showed significant, and the effects of multiple metals on FBG were attenuated in children taking total extra-school activity ≥1 h/day, and only one type of which, low or moderate & high intensity extra-school activity reached 20 min/day (Pint <0.05). For instance, each 1-fold increased As was associated with 1.41% increased FBG in overall children taking total extra-school activity <1 h/day, while that of 0.13% in those ≥1 h/day. Meanwhile, mixture of all, essential and non-essential metals were associated with increased FBG, a trend that decreased and became nonsignificant in children having certain extra-school activity, which were dominated by Mo, Se, Ti, Cd. And such relations were substantially beneficial from extra-school activity in overweight & obesity children. Accordingly, multiple essential and non-essential metals, both individual and in mixture, were positively related to FBG in children, which might be attenuated by regular physical activity.
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Glicemia , Exercício Físico , Metais , Humanos , Criança , Pré-Escolar , Feminino , Masculino , Glicemia/análise , Metais/urina , Jejum , Poluentes Ambientais/urinaRESUMO
Gain-of-function missense variants in the cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), whereas RyR2 loss-of-function missense variants cause Ca2+ release deficiency syndrome (CRDS). Recently, truncating variants in RyR2 have also been associated with ventricular arrhythmias (VAs) and sudden cardiac death. However, there are limited insights into the potential clinical relevance and in vitro functional impact of RyR2 truncating variants. We performed genetic screening of patients presenting with syncope, VAs, or unexplained sudden death and in vitro characterization of the expression and function of RyR2 truncating variants in HEK293 cells. We identified two previously unknown RyR2 truncating variants (Y4591Ter and R4663Ter) and one splice site variant predicted to result in a frameshift and premature termination (N4717 + 15Ter). These 3 new RyR2 truncating variants and a recently reported RyR2 truncating variant, R4790Ter, were generated and functionally characterized in vitro. Immunoprecipitation and immunoblotting analyses showed that all 4 RyR2 truncating variants formed heteromers with the RyR2-wildtype (WT) protein. Each of these C-terminal RyR2 truncations was non-functional and suppressed [3H]ryanodine binding to RyR2-WT and RyR2-WT mediated store overload induced spontaneous Ca2+ release activity in HEK293 cells. The expression of these RyR2 truncating variants in HEK293 cells was markedly reduced compared with that of the full-length RyR2 WT protein. Our data indicate that C-terminal RyR2 truncating variants are non-functional and can exert a dominant negative impact on the function of the RyR2 WT protein through formation of heteromeric WT/truncation complex.
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Canal de Liberação de Cálcio do Receptor de Rianodina , Taquicardia Ventricular , Humanos , Arritmias Cardíacas/genética , Cálcio/metabolismo , Células HEK293 , Mutação , Fenótipo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismoRESUMO
Objective: Patients with radioactive iodine-refractory differentiated thyroid cancer (RAIR-DTC) are often diagnosed with delay and constrained to limited treatment options. The correlation between RAI refractoriness and the underlying genetic characteristics has not been extensively studied. Methods: Adult patients with distant metastatic DTC were enrolled and assigned to undergo next-generation sequencing of a customized 26-gene panel (ThyroLead). Patients were classified into RAIR-DTC or non-RAIR groups to determine the differences in clinicopathological and molecular characteristics. Molecular risk stratification (MRS) was constructed based on the association between molecular alterations identified and RAI refractoriness, and the results were classified as high, intermediate or low MRS. Results: A total of 220 patients with distant metastases were included, 63.2% of whom were identified as RAIR-DTC. Genetic alterations were identified in 90% of all the patients, with BRAF (59.7% vs. 17.3%), TERT promoter (43.9% vs. 7.4%), and TP53 mutations (11.5% vs. 3.7%) being more prevalent in the RAIR-DTC group than in the non-RAIR group, except for RET fusions (15.8% vs. 39.5%), which had the opposite pattern. BRAF and TERT promoter are independent predictors of RAIR-DTC, accounting for 67.6% of patients with RAIR-DTC. MRS was strongly associated with RAI refractoriness (P<0.001), with an odds ratio (OR) of high to low MRS of 7.52 [95% confidence interval (95% CI), 3.96-14.28; P<0.001] and an OR of intermediate to low MRS of 3.20 (95% CI, 1.01-10.14; P=0.041). Conclusions: Molecular alterations were associated with RAI refractoriness, with BRAF and TERT promoter mutations being the predominant contributors, followed by TP53 and DICER1 mutations. MRS might serve as a valuable tool for both prognosticating clinical outcomes and directing precision-based therapeutic interventions.
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RATIONALE: Ca2+ alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca2+ alternans remains undefined. Increasing evidence suggests that Ca2+ alternans results from alternations in the inactivation of cardiac RyR2 (ryanodine receptor 2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca2+ alternans are unknown. OBJECTIVE: To determine the role of CaM (calmodulin) on Ca2+ alternans in intact working mouse hearts. METHODS AND RESULTS: We used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type, a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 wild type or mutant mouse hearts. We monitored Ca2+ transients in ventricular myocytes near the adenovirus-injection sites in Langendorff-perfused intact working hearts using confocal Ca2+ imaging. We found that CaM-wild type and CaM-M37Q promoted Ca2+ alternans and prolonged Ca2+ transient recovery in intact RyR2 wild type and mutant hearts, whereas CaM (1-4) exerted opposite effects. Altered CaM function also affected the recovery from inactivation of the L-type Ca2+ current but had no significant impact on sarcoplasmic reticulum Ca2+ content. Furthermore, we developed a novel numerical myocyte model of Ca2+ alternans that incorporates Ca2+-CaM-dependent regulation of RyR2 and the L-type Ca2+ channel. Remarkably, the new model recapitulates the impact on Ca2+ alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca2+ elevation as a result of rapid pacing triggers Ca2+-CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes sarcoplasmic reticulum Ca2+ release, which, in turn, reduces diastolic cytosolic Ca2+, leading to alternations in diastolic cytosolic Ca2+, RyR2 inactivation, and sarcoplasmic reticulum Ca2+ release (ie, Ca2+ alternans). CONCLUSIONS: Our results demonstrate that inactivation of RyR2 by Ca2+-CaM is a major determinant of Ca2+ alternans, making Ca2+-CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.
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Sinalização do Cálcio , Coração/fisiologia , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio Tipo L/metabolismo , Calmodulina/metabolismo , Células Cultivadas , Frequência Cardíaca , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica , Miócitos Cardíacos/fisiologiaRESUMO
A palladium-catalyzed dearomatization of indoles with alkynes has been developed, providing an efficient route to access a variety of synthetically useful spirocyclohexaneindolenines in moderate to good yields. The current method features a simple catalytic system, operational simplicity, and good functional group compatibility, which will contribute substantially to the development of dearomatization to access spiro compounds. Besides, the ubiquitous existence of spiro molecules, including spirocyclohexaneindolenines, in drugs and biological active molecules suggests the potential application of this methodology in medicinal chemistry.
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Evidence on joint association of a phthalate mixture with thyroid function among children and its underlying mechanism is largely unknown. We aimed to explore the associations of 10 urinary phthalate metabolites (mPAEs), either as individuals or as a mixture, with thyroid function indicators [free thyroxine, free triiodothyronine (FT3), and thyroid-stimulating hormone (TSH)] in 144 children aged 4-12 years with up to 3 repeated visits across 3 seasons. Significant and positive associations were observed for mono-(2-ethylhexyl) phthalate (MEHP), mono-iso-butyl phthalate (MiBP), and mono-n-butyl phthalate (MnBP) with TSH, as well as monobenzyl phthalate (MBzP) with FT3 in dose-response manners. The relationship between MEHP and TSH remained robust in multiple-phthalate models. Bayesian kernel machine regression (BKMR) models revealed overall linear associations of the 10 mPAE mixture with higher TSH and FT3 levels, and MEHP and MBzP were major contributors. Meanwhile, MEHP, MiBP, and MnBP were linked to the elevation of multiple cytokines including CCL 27, CCL3, CXCL1, and IL-16. Among them, IL-16 mediated the relationships of MEHP and MiBP with TSH, and the mediated proportions were 24.16% and 24.27%, respectively. Our findings suggested that mPAEs dominated by MEHP were dose-responsively associated with elevated TSH among healthy children and mediated by IL-16.
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Poluentes Ambientais , Ácidos Ftálicos , Criança , Humanos , Exposição Ambiental , Glândula Tireoide/metabolismo , Teorema de Bayes , Interleucina-16 , Ácidos Ftálicos/metabolismo , TireotropinaRESUMO
BACKGROUND: To determine whether intra-arterial thrombolysis (IAT) within 16 hours after the onset of symptoms is feasible and associated with better visual outcomes in patients with acute retinal ischemia (ARI). METHODS: The retrospective cohort study was performed from January 2014 to December 2021 in the Xuanwu Hospital of Capital Medical University. Patients with ARI who initially presented visual acuity of 20/100 or worse were screened in the study. Visual end points were evaluated at one week and at final visit after treatment. Serious adverse events were recorded during operation and within 1 week after IAT treatment. RESULTS: The amount of clinically significant visual improvement (≥0.3 logarithm of the minimum angle of resolution) in the IAT group was significantly higher than that in the conservative treatment group at one week after the treatment (47.8% vs 16.7%; P = 0.014) and at final visit (52.2% vs 20%; P = 0.014). After controlling confounding factors, ARI treatment was the only factor significantly associated with the amount of clinically significant visual improvement (OR, 4.364; 95 CI, 1.298-14.667; P = 0.017). A patient (4.3%) experienced retinal hemorrhage without symptom within 1 week after IAT treatment. No patients experienced new symptomatic cerebral infarction, intracranial hemorrhage, TIA, artery dissection, vascular perforation, and distal embolization during operation and within 1 week after IAT treatment. CONCLUSIONS: IAT may be associated with better visual improvement within 16 hours after the onset of symptoms. Besides, IAT is feasible and associated with a low risk of periprocedural complications for ARI. This study will aid in feasibility testing and sample size calculations in advance of future, fully-powered efficacy studies for ARI.
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Isquemia Encefálica , Acidente Vascular Cerebral , Humanos , Estudos Retrospectivos , Estudos de Coortes , Terapia Trombolítica , Resultado do Tratamento , Isquemia , Isquemia Encefálica/complicações , Acidente Vascular Cerebral/diagnóstico , Fibrinolíticos/uso terapêuticoRESUMO
INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.
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Esclerose Lateral Amiotrófica , Demência Frontotemporal , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Esclerose Lateral Amiotrófica/genética , Demência Frontotemporal/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína C9orf72/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sistemas CRISPR-Cas , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Neurônios/metabolismo , Expansão das Repetições de DNA/genética , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismoRESUMO
Because of the extremely polarized morphology, the proper functioning of neurons largely relies on the efficient cargo transport along the axon. Axonal transport defects have been reported in multiple neurodegenerative diseases as an early pathological feature. The discovery of mutations in human genes involved in the transport machinery provide a direct causative relationship between axonal transport defects and neurodegeneration. Here, we summarize the current genetic findings related to axonal transport in neurodegenerative diseases, and we discuss the relationship between axonal transport defects and other pathological changes observed in neurodegeneration. In addition, we summarize the therapeutic approaches targeting the axonal transport machinery in studies of neurodegenerative diseases. Finally, we review the technical advances in tracking axonal transport both in vivo and in vitro.
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Transporte Axonal/efeitos dos fármacos , Transporte Axonal/genética , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Animais , Humanos , Mutação , Doenças Neurodegenerativas/metabolismoRESUMO
Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca2+- and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca2+-independent basal activity and Ca2+-dependent activation of [3H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.
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Ativação do Canal Iônico , Mutação/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Análise Mutacional de DNA , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rianodina/metabolismo , Trítio/metabolismoRESUMO
Nucleotides metabolism is a fundamental process in all organisms. Two families of nucleoside phosphorylases (NP) that catalyze the phosphorolytic cleavage of the glycosidic bond in nucleosides have been found, including the trimeric or hexameric NP-I and dimeric NP-II family enzymes. Recent studies revealed another class of NP protein in Escherichia coli named Pyrimidine/purine nucleoside phosphorylase (ppnP), which can catalyze the phosphorolysis of diverse nucleosides and yield d-ribose 1-phosphate and the respective free bases. Here, we solved the crystal structures of ppnP from E. coli and the other three species. Our studies revealed that the structure of ppnP belongs to the RlmC-like Cupin fold and showed as a rigid dimeric conformation. Detail analysis revealed a potential nucleoside binding pocket full of hydrophobic residues, and the residues involved in the dimer and pocket formation are all well conserved in bacteria. Since the Cupin fold is a large superfamily in the biosynthesis of natural products, our studies provide the structural basis for understanding, and the directed evolution of NP proteins.
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Nucleosídeos , Purina-Núcleosídeo Fosforilase , Escherichia coli/metabolismo , Nucleosídeos/metabolismo , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Pirimidina Fosforilases/metabolismo , Pirimidinas , Especificidade por SubstratoRESUMO
Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an intracellular Ca2+ release channel critical for numerous cellular processes. Despite its ubiquitous physiological significance, ITPR1 mutations have thus far been linked to primarily movement disorders. Surprisingly, most disease-associated ITPR1 mutations generate a loss of function. This leaves our understanding of ITPR1-associated pathology oddly one-sided, as little is known about the pathological consequences of ITPR1 gain of function (GOF). To this end, we generated an ITPR1 gating domain mutation (D2594K) that substantially enhanced the inositol trisphosphate (IP3 )-sensitivity of ITPR1, and a mouse model expressing this ITPR1-D2594K+/- GOF mutation. We found that heterozygous ITPR1-D2594K+/- mutant mice exhibited male infertility, azoospermia, and acrosome loss. Furthermore, we functionally characterized a human ITPR1 variant V494I identified in the UK Biobank database as potentially associated with disorders of the testis. We found that the ITPR1-V494I variant significantly enhanced IP3 -induced Ca2+ release in HEK293 cells. Thus, ITPR1 hyperactivity may increase the risk of testicular dysfunction.
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Mutação com Ganho de Função , Infertilidade Masculina , Receptores de Inositol 1,4,5-Trifosfato , Animais , Cálcio/metabolismo , Células HEK293 , Humanos , Infertilidade Masculina/genética , Inositol 1,4,5-Trifosfato , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Camundongos , Mutação/genéticaRESUMO
Significant advances have been achieved for the construction of chiral skeletons containing 1,2,3-triazoles via transition-metal-catalyzed asymmetric azide-alkyne cycloaddition; however, most of them have been limited to terminal alkynes in the synthesis of central chirality via desymmetrization and dynamic/dynamic kinetic resolution. Enantioselective transition-metal-catalyzed azide-internal-alkyne cycloaddition is extremely limited. Moreover, the construction of a challenging five-membered (hetero)biaryl axially chiral molecule via transition-metal-catalyzed asymmetric azide-internal-alkyne cycloaddition is still underexplored. Herein, we first report an atroposelective and atom-economical synthesis of axially chiral 1,4,5-trisubstituted 1,2,3-triazoles, directly acting as core chiral units of challenging five-membered atropisomers, via the enantioselective Rh-catalyzed azide-alkyne cycloaddition (E-RhAAC) of internal alkynes and azides. The reaction demonstrates excellent functional group tolerance, forging a variety of C-C axially chiral 1,2,3-triazoles under mild conditions with moderate to excellent yields (up to 99% yield) and generally high to excellent enantioselectivities (up to 99% ee) along with specific regiocontrol. The origin of regio- and enantioselectivity control is disclosed by density functional theory (DFT) calculations, providing new guidance for the facile construction of axially chiral compounds.
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Azidas , Ródio , Alcinos , Catálise , Reação de Cicloadição , Estereoisomerismo , TriazóisRESUMO
Energy deprivation and reduced levels of hydrogen sulfide (H2S) in the brain is closely associated with Alzheimer's disease (AD). However, there is currently no fluorescent probe for precise exploration of both H2S and adenosine triphosphate (ATP) to directly demonstrate their relationship and their dynamic pattern changes. Herein, we developed a two-photon fluorescent probe, named AD-3, to simultaneously image endogenous H2S and ATP from two emission channels of fluorescent signals in live rat brains with AD. The probe achieved excellent selectivity and good detection linearity for H2S in the 0-100 µM concentration range and ATP in the 2-5 mM concentration range, respectively, with a detection limit of 0.19 µM for H2S and 0.01 mM for ATP. Fluorescence imaging in live cells reveals that such probe could successfully apply for simultaneous imaging and accurate quantification of H2S and ATP in neuronal cells. Further using real-time quantitative polymerase chain reaction and Western blots, we confirmed that H2S regulates ATP synthesis by acting on cytochrome C, cytochrome oxidase subunit 3 of complex IV, and protein 6 of complex I in the mitochondrial respiratory chain. Subsequently, we constructed a high-throughput screening platform based on AD-3 probe to rapidly screen the potential anti-AD drugs to control glutamate-stimulated oxidative stress associated with abnormal H2S and ATP levels. Significantly, AD-3 probe was found capable of imaging of H2S and ATP in APP/PS1 mice, and the concentration of H2S and ATP in the AD mouse brain was found to be lower than that in wild-type mice.
Assuntos
Doença de Alzheimer , Sulfeto de Hidrogênio , Trifosfato de Adenosina , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Animais , Corantes Fluorescentes , Ácido Glutâmico , Células HeLa , Humanos , Sulfeto de Hidrogênio/análise , Camundongos , Fótons , RatosRESUMO
The link between phthalates exposure and arterial stiffness in adults remains unclear. We aimed to investigate the associations of urinary phthalate metabolites with arterial stiffness in a longitudinal panel study involving 3 repeated visits among 127 Chinese adults. Urine samples were collected once a day for 4 consecutive days and 10 urinary phthalate metabolites were measured by gas chromatography-tandem mass spectrometry (GC-MS/MS). Brachial ankle pulse wave velocity (baPWV) and ankle-brachial index (ABI) were determined using an oscillometric device (BP-203RPEIII; Omron) in physical examinations during each visit. Linear mixed-effect (LME) models with the adaptive Least Absolute Shrinkage and Selection Operator (LASSO) method were applied to assess the associations between urinary phthalate metabolites and arterial stiffness parameters. The odds ratio (OR) for peripheral arterial disease (PAD) was estimated using generalized estimating equations. For ABI, mono-methyl phthalate (MMP) and mono-n-butyl phthalate (MBP) at lag 0 day were selected by the adaptive LASSO, whereas no phthalates were selected for baPWV. After adjusting for potential covariates and other metabolites, we found ABI reduction was associated with one-unit increase of ln-transformed urinary MBP at lag 0 day [ß = 0.013 (SE = 0.006), P = 0.003)]. Stratified analysis revealed that the inverse association was more evident in males (Pinteraction = 0.025). In addition, we observed a borderline risk of PAD in relation to MBP exposure at lag 0 day (P = 0.06). Our data suggested that environmental exposure to MBP may contribute to arterial stiffness, and the effect seems to be sex-specific.
Assuntos
Ácidos Ftálicos , Rigidez Vascular , Índice Tornozelo-Braço , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Masculino , Ácidos Ftálicos/urina , Análise de Onda de Pulso , Espectrometria de Massas em TandemRESUMO
Studies on associations of metals with leucocyte telomere length (LTL) were mainly limited to several most common toxic metals and single-metal effect, but the impact of other common metals and especially the overall joint associations and interactions of metal mixture with LTL are largely unknown. We included 15 plasma metals and LTL among 4906 participants from Dongfeng-Tongji cohort. Multivariable linear regression was used to estimate associations of individual metals with LTL. We also applied Bayesian kernel machine regression (BKMR) and quantile g-computation regression (Q-g) to evaluate the overall association and interactions, and identified the major contributors as well as the potential modifications by major characteristics. Multivariable linear regression found vanadium, copper, arsenic, aluminum and nickel were negatively associated with LTL, and a 2-fold change was related to 1.9%-5.1% shorter LTL; while manganese and zinc showed 3.7% and 4.0% longer LTL (all P < 0.05) in multiple-metal models. BKMR confirmed above metals and revealed a linearly inverse joint association between 15 metals and LTL. Q-g regression further indicated each quantile increase in mixture was associated with 5.2% shorter LTL (95% CI: -8.1%, -2.3%). Furthermore, manganese counteracted against aluminum and vanadium respectively (Pint<0.05). In addition, associations of vanadium, aluminum and metal mixture with LTL were more prominent in overweight participants. Our results are among the first to provide a new comprehensive view of metal mixture exposure on LTL attrition in the general population, including identifying the major components, metals interactions and the overall effects.