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Generation of genetically uniform non-human primates may help to establish animal models for primate biology and biomedical research. In this study, we have successfully cloned cynomolgus monkeys (Macaca fascicularis) by somatic cell nuclear transfer (SCNT). We found that injection of H3K9me3 demethylase Kdm4d mRNA and treatment with histone deacetylase inhibitor trichostatin A at one-cell stage following SCNT greatly improved blastocyst development and pregnancy rate of transplanted SCNT embryos in surrogate monkeys. For SCNT using fetal monkey fibroblasts, 6 pregnancies were confirmed in 21 surrogates and yielded 2 healthy babies. For SCNT using adult monkey cumulus cells, 22 pregnancies were confirmed in 42 surrogates and yielded 2 babies that were short-lived. In both cases, genetic analyses confirmed that the nuclear DNA and mitochondria DNA of the monkey offspring originated from the nucleus donor cell and the oocyte donor monkey, respectively. Thus, cloning macaque monkeys by SCNT is feasible using fetal fibroblasts.
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Clonagem de Organismos , Técnicas de Transferência Nuclear , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Feminino , Ácidos Hidroxâmicos/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/metabolismo , Macaca fascicularis , GravidezRESUMO
Insulin resistance is a hallmark of diabetes and an unmet clinical need. Insulin inhibits hepatic glucose production and promotes lipogenesis by suppressing FOXO1-dependent activation of G6pase and inhibition of glucokinase, respectively. The tight coupling of these events poses a dual conundrum: mechanistically, as the FOXO1 corepressor of glucokinase is unknown, and clinically, as inhibition of glucose production is predicted to increase lipogenesis. Here, we report that SIN3A is the insulin-sensitive FOXO1 corepressor of glucokinase. Genetic ablation of SIN3A abolishes nutrient regulation of glucokinase without affecting other FOXO1 target genes and lowers glycemia without concurrent steatosis. To extend this work, we executed a small-molecule screen and discovered selective inhibitors of FOXO-dependent glucose production devoid of lipogenic activity in hepatocytes. In addition to identifying a novel mode of insulin action, these data raise the possibility of developing selective modulators of unliganded transcription factors to dial out adverse effects of insulin sensitizers.
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Proteína Forkhead Box O1/antagonistas & inibidores , Glucose/metabolismo , Hepatócitos/metabolismo , Resistência à Insulina , Acetilação , Animais , Células Cultivadas , Proteína Forkhead Box O1/química , Glucoquinase/genética , Glucoquinase/metabolismo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Células HEK293 , Hepatócitos/enzimologia , Histona Desacetilases/metabolismo , Humanos , Lipogênese/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fosforilação , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
TrkB (neuronal receptor tyrosine kinase-2, NTRK2) is the receptor for brain-derived neurotrophic factor (BDNF) and is a critical regulator of activity-dependent neuronal plasticity. The past few years have witnessed an increasing understanding of the structure and function of TrkB, including its transmembrane domain (TMD). TrkB interacts with membrane cholesterol, which bidirectionally regulates TrkB signaling. Additionally, TrkB has recently been recognized as a binding target of antidepressant drugs. A variety of different antidepressants, including typical and rapid-acting antidepressants, as well as psychedelic compounds, act as allosteric potentiators of BDNF signaling through TrkB. This suggests that TrkB is the common target of different antidepressant compounds. Although more research is needed, current knowledge suggests that TrkB is a promising target for further drug development.
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Glicoproteínas de Membrana , Receptor trkB , Humanos , Receptor trkB/metabolismo , Receptor trkB/química , Animais , Domínios Proteicos , Transdução de Sinais , Antidepressivos/uso terapêutico , Antidepressivos/farmacologia , Antidepressivos/química , Antidepressivos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/químicaRESUMO
Eukaryotic cells learn and adapt via unknown network architectures. Recent work demonstrated a circuit of two GTPases used by cells to overcome growth factor scarcity, encouraging our view that artificial and biological intelligence share strikingly similar design principles and that cells function as deep reinforcement learning (RL) agents in uncertain environments.
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GTP Fosfo-Hidrolases , Transdução de Sinais , GTP Fosfo-Hidrolases/metabolismoRESUMO
Alzheimer's disease (AD) pathogenesis has been linked to the accumulation of longer, aggregation-prone amyloid ß (Aß) peptides in the brain. Γ-secretases generate Aß peptides from the amyloid precursor protein (APP). Γ-secretase modulators (GSMs) promote the generation of shorter, less-amyloidogenic Aßs and have therapeutic potential. However, poorly defined drug-target interactions and mechanisms of action have hampered their therapeutic development. Here, we investigate the interactions between the imidazole-based GSM and its target γ-secretase-APP using experimental and in silico approaches. We map the GSM binding site to the enzyme-substrate interface, define a drug-binding mode that is consistent with functional and structural data, and provide molecular insights into the underlying mechanisms of action. In this respect, our analyses show that occupancy of a γ-secretase (sub)pocket, mediating binding of the modulator's imidazole moiety, is sufficient to trigger allosteric rearrangements in γ-secretase as well as stabilize enzyme-substrate interactions. Together, these findings may facilitate the rational design of new modulators of γ-secretase with improved pharmacological properties.
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Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Humanos , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Inibidores e Moduladores de Secretases gama , Doença de Alzheimer/metabolismo , Imidazóis/uso terapêuticoRESUMO
The recently developed double-click reaction sequence [G. Meng et al., Nature 574, 86-89 (2019)] is expected to vastly expand the number and diversity of synthetically accessible 1,2,3-triazole derivatives. However, it remains elusive how to rapidly navigate the extensive chemical space created by double-click chemistry for bioactive compound discovery. In this study, we selected a particularly challenging drug target, the glucagon-like-peptide-1 receptor (GLP-1R), to benchmark our new platform for the design, synthesis, and screening of double-click triazole libraries. First, we achieved a streamlined synthesis of customized triazole libraries on an unprecedented scale (composed of 38,400 new compounds). By interfacing affinity-selection mass spectrometry and functional assays, we identified a series of positive allosteric modulators (PAMs) with unreported scaffolds that can selectively and robustly enhance the signaling activity of the endogenous GLP-1(9-36) peptide. Intriguingly, we further revealed an unexpected binding mode of new PAMs which likely act as a molecular glue between the receptor and the peptide agonist. We anticipate the merger of double-click library synthesis with the hybrid screening platform allows for efficient and economic discovery of drug candidates or chemical probes for various therapeutic targets.
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Receptor do Peptídeo Semelhante ao Glucagon 1 , Peptídeos , Regulação Alostérica , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Peptídeos/química , Triazóis/químicaRESUMO
Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of ß-catenin by CRL1ß-TrCP and Nedd8-CRL1ß-TrCP almost equally. Thus, neddylation of CRL1ß-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.
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Ligantes , Ubiquitina-Proteína Ligases , Ubiquitinação , Animais , Humanos , Camundongos , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas Culina/metabolismo , Suramina/farmacologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Proteína NEDD8/metabolismoRESUMO
Transient receptor potential canonical (TRPC) proteins assemble to form homo- or heterotetrameric, nonselective cation channels permeable to K+, Na+, and Ca2+. TRPC channels are thought to act as complex integrators of physical and chemical environmental stimuli. Although the understanding of essential physiological roles of TRPC channels is incomplete, their implication in various pathological mechanisms and conditions of the nervous system, kidneys, and cardiovascular system in combination with the lack of major adverse effects of TRPC knockout or TRPC channel inhibition is driving the search of TRPC channel modulators as potential therapeutics. Here, we review the most promising small-molecule TRPC channel modulators, the understanding of their mode of action, and their potential in the study and treatment of cardiovascular and metabolic disease.
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Sistema Cardiovascular , Canais de Potencial de Receptor Transitório , Sistema Cardiovascular/metabolismo , Humanos , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Cystic fibrosis (CF) is an inherited multisystemic disease that can cause progressive bronchiectasis, pancreatic endocrine and exocrine insufficiency, distal intestinal obstruction syndrome, liver dysfunction, and other disorders. Traditional therapies focused on the treatment or prevention of damage to each organ system with incremental modalities such as nebulized medications for the lungs, insulin for diabetes, and supplementation with pancreatic enzymes. However, the advent of highly effective modulator therapies that target specific cystic fibrosis transmembrane conductance regulator protein malformations resulting from individual genetic mutations has transformed the lives and prognosis for persons with CF.
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Fibrose Cística , Diabetes Mellitus , Humanos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Prognóstico , Mutação , Aminofenóis/uso terapêuticoRESUMO
The genome-wide perturbation of transcriptional networks with CRISPR-Cas technology has primarily involved systematic and targeted gene modulation. Here, we developed PRISM (Perturbing Regulatory Interactions by Synthetic Modulators), a screening platform that uses randomized CRISPR-Cas transcription factors (crisprTFs) to globally perturb transcriptional networks. By applying PRISM to a yeast model of Parkinson's disease (PD), we identified guide RNAs (gRNAs) that modulate transcriptional networks and protect cells from alpha-synuclein (αSyn) toxicity. One gRNA identified in this screen outperformed the most protective suppressors of αSyn toxicity reported previously, highlighting PRISM's ability to identify modulators of important phenotypes. Gene expression profiling revealed genes differentially modulated by this strong protective gRNA that rescued yeast from αSyn toxicity when overexpressed. Human homologs of top-ranked hits protected against αSyn-induced cell death in a human neuronal PD model. Thus, high-throughput and unbiased perturbation of transcriptional networks via randomized crisprTFs can reveal complex biological phenotypes and effective disease modulators.
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Sistemas CRISPR-Cas , Redes Reguladoras de Genes , RNA Guia de Cinetoplastídeos/genética , Fatores de Transcrição/genética , Transcrição Gênica , alfa-Sinucleína/genética , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Modelos Biológicos , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fenótipo , RNA Guia de Cinetoplastídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transgenes , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/metabolismoRESUMO
The mammalian cryptochrome isoforms, CRY1 and CRY2, are core circadian clock regulators that work redundantly. Recent studies revealed distinct roles of these closely related homologs in clock output pathways. Isoform-selective control of CRY1 and CRY2 is critical for further understanding their redundant and distinct roles. KL001 was the first identified small-molecule CRY modulator that activates both CRY1 and CRY2. SHP656 is an orally available KL001 derivative and has shown efficacy in blood glucose control and inhibition of glioblastoma stem cell (GSC) growth in animal models. However, CRY isoform selectivity of SHP656 was uncharacterized, limiting understanding of the roles of CRY1 and CRY2. Here, we report the elucidation of CRY2 selectivity of SHP656. SHP656 lengthened cellular circadian period in a CRY2-dependent manner and selectively interacted with CRY2. By determining the X-ray crystal structure of CRY2 in complex with SHP656 and performing molecular dynamics simulations, we elucidated compound interaction mechanisms. SHP656 binding was compatible with the intrinsic CRY2 gatekeeper W417 "in" orientation and also a close "further in" conformation. Perturbation of W417 interaction with the lid loop resulted in a reduced effect of SHP656 on CRY2, supporting an important role of gatekeeper orientation in isoform selectivity. We also identified the R form of SHP656 (called SHP1703) as the active isomer. Treatment with SHP1703 effectively reduced GSC viability. Our results suggest a direct role of CRY2 in glioblastoma antitumorigenesis and provide a rationale for the selective modulation of CRY isoforms in the therapeutic treatment of glioblastoma and other circadian clock-related diseases.
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Relógios Circadianos , Glioblastoma , Animais , Carbazóis , Ritmo Circadiano/fisiologia , Criptocromos/metabolismo , Glioblastoma/tratamento farmacológico , Mamíferos/metabolismo , Isoformas de Proteínas/genética , SulfonamidasRESUMO
Electrical neuron stimulation holds promise for treating chronic neurological disorders, including spinal cord injury, epilepsy, and Parkinson's disease. The implementation of ultrathin, flexible electrodes that can offer noninvasive attachment to soft neural tissues is a breakthrough for timely, continuous, programable, and spatial stimulations. With strict flexibility requirements in neural implanted stimulations, the use of conventional thick and bulky packages is no longer applicable, posing major technical issues such as short device lifetime and long-term stability. We introduce herein a concept of long-lived flexible neural electrodes using silicon carbide (SiC) nanomembranes as a faradic interface and thermal oxide thin films as an electrical barrier layer. The SiC nanomembranes were developed using a chemical vapor deposition (CVD) process at the wafer level, and thermal oxide was grown using a high-quality wet oxidation technique. The proposed material developments are highly scalable and compatible with MEMS technologies, facilitating the mass production of long-lived implanted bioelectrodes. Our experimental results showed excellent stability of the SiC/silicon dioxide (SiO2) bioelectronic system that can potentially last for several decades with well-maintained electronic properties in biofluid environments. We demonstrated the capability of the proposed material system for peripheral nerve stimulation in an animal model, showing muscle contraction responses comparable to those of a standard non-implanted nerve stimulation device. The design concept, scalable fabrication approach, and multimodal functionalities of SiC/SiO2 flexible electronics offer an exciting possibility for fundamental neuroscience studies, as well as for neural stimulation-based therapies.
Assuntos
Terapia por Estimulação Elétrica , Neuroestimuladores Implantáveis , Nanoestruturas , Semicondutores , Compostos Inorgânicos de Carbono/química , Terapia por Estimulação Elétrica/instrumentação , Membranas Artificiais , Compostos de Silício/química , Dióxido de Silício/químicaRESUMO
BACKGROUND: Pseudomonas aeruginosa is a multidrug-resistant pathogen causing recalcitrant pulmonary infections in people with cystic fibrosis (pwCF). Cystic fibrosis transmembrane conductance regulator (CFTR) modulators have been developed that partially correct the defective chloride channel driving disease. Despite the many clinical benefits, studies in adults have demonstrated that while P. aeruginosa sputum load decreases, chronic infection persists. Here, we investigate how P. aeruginosa in pwCF may change in the altered lung environment after CFTR modulation. METHODS: P. aeruginosa strains (n = 105) were isolated from the sputum of 11 chronically colonized pwCF at baseline and up to 21 months posttreatment with elexacaftor-tezacaftor-ivacaftor or tezacaftor-ivacaftor. Phenotypic characterization and comparative genomics were performed. RESULTS: Clonal lineages of P. aeruginosa persisted after therapy, with no evidence of displacement by alternative strains. We identified commonly mutated genes among patient isolates that may be positively selected for in the CFTR-modulated lung. However, classic chronic P. aeruginosa phenotypes such as mucoid morphology were sustained, and isolates remained just as resistant to clinically relevant antibiotics. CONCLUSIONS: Despite the clinical benefits of CFTR modulators, clonal lineages of P. aeruginosa persist that may prove just as difficult to manage in the future, especially in pwCF with advanced lung disease.
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We broaden the clinical versatility of human nasal epithelial (HNE) cells. HNEs were isolated from 10 participants harboring CFTR variants: nine with rare variants (Q359R [n=2], G480S, R334W [n=5], and R560T) and one person harboring R117H;7T;TG10/5T;TG12. Cultures were differentiated at air-liquid interface. CFTR function was measured in Ussing chambers at three conditions - baseline, ivacaftor, and elexacaftor+tezacaftor+ivacaftor (ETI). Four participants initiated modulators. Q359R HNEs had 5.4% (%WT) baseline CFTR function and 25.5% with ivacaftor. With therapy, sweat [Cl-] decreased and symptoms resolved. G480S HNEs had 4.1% baseline and 32.1% CFTR function with ETI. Clinically, FEV1 increased and sweat [Cl-] decreased (119 to 46mmol/L) with ETI. In vitro cultures derived from five individuals harboring R334W showed a moderate increase in CFTR function with exposure to modulators. For one of these participants, ETI was begun in vivo; symptoms and FEV1 improved. c.1679G>C (R560T) HNEs had <4% baseline CFTR function and no modulator response. RNA analysis confirmed that c.1679G>C completely mis-splices. A symptomatic patient harboring R117H;7T;TG10/5T;TG12 exhibited reduced CFTR function (17.5%) in HNEs, facilitating mild CF diagnosis. HNEs responded to modulators (ivacaftor: 32.8%, ETI: 55.5%) and, since beginning therapy, lung function improved. While reaffirming HNE use for guiding therapeutic approaches, we inform predictions on modulator response (e.g. R334W) and closely assess variants affecting splicing (e.g. c.1679G>C). Notably, functional studies in HNEs harboring R117H;7T;TG10/5T;TG12 facilitated mild CF diagnosis, suggesting use for HNE functional studies as a clinical diagnostic test.
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Elexacaftor/tezacaftor/ivacaftor (ETI) has made a substantial positive impact for people living with CF (pwCF). However, there can be substantial variability in efficacy, and we lack adequate biomarkers to predict individual response. We thus aimed to identify transcriptomic profiles in nasal respiratory epithelium that predict clinical response to ETI treatment. We obtained nasal epithelial samples from pwCF prior to ETI initiation and performed a transcriptome-wide analysis of baseline gene expression to predict changes in FEV1 (∆FEV1), year's best FEV1 (∆ybFEV1), and body mass index (∆BMI). Using the top differentially expressed genes (DEGs), we generated transcriptomic risk scores (TRS) and evaluated their predictive performance. The study included 40 pwCF aged ≥6 years (mean 27.7 [SD=15.1] years; 40% female). After ETI initiation, FEV1 improved ≥5% in 22 (61.1%) participants and ybFEV1 improved ≥5% in 19 (50%). TRS were constructed using top over-expressed and under-expressed genes for each. Adding the ∆FEV1 TRS for to a model with age, sex, and baseline FEV1 increased the AUC from 0.41 to 0.88; the ∆ybFEV1 TRS increased the AUC from 0.51 to 0.88; and the ∆BMI TRS increased the AUC from 0.46 to 0.92. Average accuracy was thus ~85% in predicting the response to the three outcomes. Results were similar in models further adjusted for F508del zygosity and previous CFTR modulator use. In conclusion, we identified nasal epithelial transcriptomic profiles that help accurately predict changes in FEV1 and BMI with ETI treatment. These novel TRS could serve as predictive biomarkers for clinical response to modulator treatment in pwCF.
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Arising out of a PhD project more than 50 years ago to synthesise analogues of the neurotransmitter GABA, a series of new chemical entities were found to have selective actions on ionotropic GABA receptors. Several of these neurochemicals are now commercially available. A new subtype of these receptors was discovered that could be a target for the treatment of myopia, the facilitation of learning and memory, and the improvement of post-stroke motor recovery. The development of these new chemical entities over many years demonstrates the importance of neurochemicals with which to investigate selective aspects of GABA receptors and illustrates the significance of collaboration between chemists and biologists in neurochemistry. Vital were the improvements in synthetic organic chemistry and the use of functional human receptors expressed in oocytes. Current interest in ionotropic GABA receptors includes the clinical development of subtype-specific agents and the role of gain-of-function receptor variants in epilepsy. Dietary flavonoids were found to cross the blood-brain barrier to influence brain function. Natural and synthetic flavonoids had a range of effects on GABA receptors, ranging from positive, silent, and negative allosteric modulators, to even second-order modulation of first-order modulators. Flavonoids have been called "a new family of benzodiazepines." Like benzodiazepines, flavonoids reduce stress. Stress produces changes in GABA receptors in the brain that may be because of changes in endogenous modulators, such as neurosteroids and corticosteroids. GABA also occurs naturally in the diet leading to studies of the effects of oral GABA on brain function. This finding has resulted in studies of GABA and related neurochemicals as neuro-nutraceuticals. GABA systems in the gut microbiome are essential to such studies. The actions of oral GABA and of GABA-enriched beverages and foodstuffs are now an area of considerable scientific and commercial interest. GABA is a deceptively simple chemical that can take up many shapes, which may underlie its complex functions. The need for new chemical entities with selective actions for further studies highlights the need for continuing collaboration between chemists and biologists.
Assuntos
Epilepsia , Flavonoides , Receptores de GABA , Ácido gama-Aminobutírico , Humanos , Animais , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Ácido gama-Aminobutírico/metabolismo , Receptores de GABA/metabolismo , Receptores de GABA/efeitos dos fármacos , Flavonoides/farmacologia , Flavonoides/química , Flavonoides/uso terapêutico , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/metabolismo , Moduladores GABAérgicos/farmacologiaRESUMO
Cholinesterase enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are traditionally associated with the termination of acetylcholine mediated neural signaling. The fact that these ubiquitous enzymes are also found in tissues not involved in neurotransmission has led to search for alternative functions for these enzymes. Cholinesterases are reported to be involved in many lipid related disease states. Taking into view that lipases and cholinesterases belong to the same enzyme class and by comparing the catalytic sites, we propose a new outlook on the link between BChE and lipid metabolism. The lipogenic substrates of BChE that have recently emerged in contrast to traditional cholinesterase substrates are explained through the hydrolytic capacity of BChE for ghrelin, 4-methyumbelliferyl (4-mu) palmitate, and arachidonoylcholine and through endogenous lipid mediators such as cannabinoids like anandamide and essential fatty acids. The abundance of BChE in brain, intestine, liver, and plasma, tissues with active lipid metabolism, supports the idea that BChE may be involved in lipid hydrolysis. BChE is also regulated by various lipids such as linoleic acid, alpha-linolenic acid or dioctanoylglycerol, whereas AChE is inhibited. The finding that BChE is able to hydrolyze 4-mu palmitate at a pH where lipases are less efficient points to its role as a backup in lipolysis. In diseases such as Alzheimer, in which elevated BChE and impaired lipid levels are observed, the lipolytic activity of BChE might be involved. It is possible to suggest that fatty acids such as 4-mu palmitate, ghrelin, arachidonoylcholine, essential fatty acids, and other related lipid mediators regulate cholinesterases, which could lead to some sort of compensatory mechanism at high lipid concentrations.
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Butirilcolinesterase , Metabolismo dos Lipídeos , Acetilcolinesterase/metabolismo , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Ácidos Graxos , Grelina/metabolismo , Ácido Linoleico , Metabolismo dos Lipídeos/genética , PalmitatosRESUMO
Activation of oncogenes to sustain proliferative signaling and initiate metastasis are important hallmarks of cancer. Oncogenes are amplified or overexpressed in cancer cells and overexpression is often controlled at the level of transcription. Gene expression is tightly controlled by many cis-regulatory elements and trans-acting factors. Large clusters of enhancers known as "super-enhancers" drive robust expression of cell-fate determining transcription factors in cell identity. Cancer cells can take advantage of super-enhancers and become transcriptionally addicted to them leading to tumorigenesis and metastasis. Additionally, the cis-regulatory landscape of cancer includes aberrant super-enhancers that are not present in normal cells. The landscape of super-enhancers in cancer is characterized by high levels of histone H3K27 acetylation and bromodomain-containing protein 4 (BRD4), and Mediator complex. These chromatin features facilitate the identification of cancer type-specific and cell-type-specific super-enhancers that control the expression of important oncogenes to stimulate their growth. Disruption of super-enhancers via inhibiting BRD4 or other epigenetic proteins is a potential therapeutic option. Here, we will describe the discovery of super-enhancers and their unique characteristics compared to typical enhancers. Then, we will highlight how super-enhancer-associated genes contribute to cancer progression in different solid tumor types. Lastly, we will cover therapeutic targets and their epigenetic modulators.
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Neoplasias , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Elementos Facilitadores Genéticos , Neoplasias/genética , Neoplasias/terapia , Cromatina , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
The activation of yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double-edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD-responsive DNA element (YRE) and incorporated it into a lentivirus-based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity in vitro and Venus fluorescence in vivo. Furthermore, by replacing YRE with TCF- and NF-κB-binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/ß-catenin/TCF- and IL-1ß/NF-κB-mediated signaling, respectively. The practicality of this system was evaluated by performing cell-based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter-introduced cells capable of quantifying YAP1/TAZ- and ß-catenin-mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet-derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts.
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The most common CFTR mutant in cystic fibrosis (CF), ΔF508 CFTR, is eliminated by ubiquitination even in the presence of CF drugs, reducing their therapeutic efficacy. RFFL is one of the ubiquitin ligases that remove ΔF508 CFTR from the cell surface despite treatment with the triple combination of CFTR modulators (TEZ/ELX/IVA) used clinically. Although RFFL knockdown has been shown to enhance the efficacy of TEZ/ELX/IVA in cell culture models, its impact in mouse models has not been evaluated. Here, we demonstrate that RFFL ablation significantly improves the effect of TEZ/ELX/IVA, resulting in enhanced function of ΔF508 CFTR in mouse organoids. Since RFFL knockout mice showed no significant abnormalities, our findings support RFFL inhibition as a promising strategy to improve CFtreatment.