RESUMEN
Single-cell sequencing methods have enabled in-depth analysis of the diversity of cell types and cell states in a wide range of organisms. These tools focus predominantly on sequencing the genomes1, epigenomes2 and transcriptomes3 of single cells. However, despite recent progress in detecting proteins by mass spectrometry with single-cell resolution4, it remains a major challenge to measure translation in individual cells. Here, building on existing protocols5-7, we have substantially increased the sensitivity of these assays to enable ribosome profiling in single cells. Integrated with a machine learning approach, this technology achieves single-codon resolution. We validate this method by demonstrating that limitation for a particular amino acid causes ribosome pausing at a subset of the codons encoding the amino acid. Of note, this pausing is only observed in a sub-population of cells correlating to its cell cycle state. We further expand on this phenomenon in non-limiting conditions and detect pronounced GAA pausing during mitosis. Finally, we demonstrate the applicability of this technique to rare primary enteroendocrine cells. This technology provides a first step towards determining the contribution of the translational process to the remarkable diversity between seemingly identical cells.
Asunto(s)
Ciclo Celular/genética , Codón/genética , Biosíntesis de Proteínas , RNA-Seq/métodos , Ribosomas/metabolismo , Análisis de la Célula Individual , Aminoácidos/deficiencia , Aminoácidos/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular , Femenino , Humanos , Aprendizaje Automático , Masculino , Ratones , Extensión de la Cadena Peptídica de Translación , Iniciación de la Cadena Peptídica Traduccional , Terminación de la Cadena Péptídica Traduccional , Biosíntesis de Proteínas/efectos de los fármacos , Reproducibilidad de los Resultados , Ribosomas/efectos de los fármacosRESUMEN
In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.
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Aminoácidos/farmacología , Compuestos de Bifenilo/farmacología , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidores , Sitio Alostérico , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Análisis Mutacional de ADN , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Enzimas Ubiquitina-Conjugadoras , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
Congenital hyperinsulinism of infancy (CHI) can be caused by a deficiency of the ubiquitously expressed enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD). To test the hypothesis that SCHAD-CHI arises from a specific defect in pancreatic ß-cells, we created genetically engineered ß-cell-specific (ß-SKO) or hepatocyte-specific (L-SKO) SCHAD knockout mice. While L-SKO mice were normoglycemic, plasma glucose in ß-SKO animals was significantly reduced in the random-fed state, after overnight fasting, and following refeeding. The hypoglycemic phenotype was exacerbated when the mice were fed a diet enriched in leucine, glutamine, and alanine. Intraperitoneal injection of these three amino acids led to a rapid elevation in insulin levels in ß-SKO mice compared to controls. Consistently, treating isolated ß-SKO islets with the amino acid mixture potently enhanced insulin secretion compared to controls in a low-glucose environment. RNA sequencing of ß-SKO islets revealed reduced transcription of ß-cell identity genes and upregulation of genes involved in oxidative phosphorylation, protein metabolism, and Ca2+ handling. The ß-SKO mouse offers a useful model to interrogate the intra-islet heterogeneity of amino acid sensing given the very variable expression levels of SCHAD within different hormonal cells, with high levels in ß- and δ-cells and virtually absent α-cell expression. We conclude that the lack of SCHAD protein in ß-cells results in a hypoglycemic phenotype characterized by increased sensitivity to amino acid-stimulated insulin secretion and loss of ß-cell identity.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasa , Aminoácidos , Hiperinsulinismo Congénito , Hipoglucemia , Secreción de Insulina , Células Secretoras de Insulina , Animales , Ratones , Aminoácidos/metabolismo , Aminoácidos/farmacología , Hipoglucemia/enzimología , Hipoglucemia/genética , Insulina/metabolismo , Secreción de Insulina/efectos de los fármacos , Ratones Noqueados , 3-Hidroxiacil-CoA Deshidrogenasa/deficiencia , 3-Hidroxiacil-CoA Deshidrogenasa/genética , Células Secretoras de Insulina/enzimología , Hiperinsulinismo Congénito/genéticaRESUMEN
Every year, the overprescription, misuse, and improper disposal of antibiotics have led to the rampant development of drug-resistant pathogens and, in turn, a significant increase in the number of patients who die of drug-resistant fungal infections. Recently, researchers have begun investigating the use of antimicrobial peptides (AMPs) as next-generation antifungal agents to inhibit the growth of drug-resistant fungi. The antifungal activity of alpha-helical peptides designed using the cationic amino acids containing lysine and arginine and the hydrophobic amino acids containing isoleucine and tryptophan were evaluated using 10 yeast and mold fungi. Among these peptides, WIK-14, which is composed of a 14-mer with tryptophan sequences at the amino terminus, showed the best antifungal activity via transient pore formation and ROS generation. In addition, the in vivo antifungal effects of WIK-14 were investigated in a mouse model infected with drug-resistant Candida albicans. The results demonstrate the potential of AMPs as antifungal agents.
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Antifúngicos , Triptófano , Ratones , Animales , Humanos , Antifúngicos/farmacología , Antifúngicos/química , Triptófano/química , Lisina/química , Péptidos Antimicrobianos , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Aminoácidos/farmacología , Candida albicans , Arginina/química , Pruebas de Sensibilidad MicrobianaRESUMEN
Learning where to find nutrients while at the same time avoiding toxic food is essential for survival of any animal. Using Drosophila melanogaster larvae as a study case, we investigate the role of gustatory sensory neurons expressing IR76b for associative learning of amino acids, the building blocks of proteins. We found surprising complexity in the neuronal underpinnings of sensing amino acids, and a functional division of sensory neurons. We found that the IR76b receptor is dispensable for amino acid learning, whereas the neurons expressing IR76b are specifically required for the rewarding but not the punishing effect of amino acids. This unexpected dissociation in neuronal processing of amino acids for different behavioural functions provides a study case for functional divisions of labour in gustatory systems.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Animales , Aminoácidos/metabolismo , Aminoácidos/farmacología , Drosophila melanogaster/fisiología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/farmacología , Neuronas/metabolismo , Recompensa , Gusto/fisiologíaRESUMEN
LY-404,039 is an orthosteric agonist at metabotropic glutamate 2 and 3 (mGlu 2/3 ) receptors, with a possible additional agonist effect at dopamine D 2 receptors. LY-404,039 and its pro-drug, LY-2140023, have previously been tested in clinical trials for psychiatric indications and could therefore be repurposed if they were shown to be efficacious in other conditions. We have recently demonstrated that the mGlu 2/3 orthosteric agonist LY-354,740 alleviated L-3,4-dihydroxyphenylalanine (L-DOPA)-induced abnormal involuntary movements (AIMs) in the 6-hydroxydopamine (6-OHDA)-lesioned rat without hampering the anti-parkinsonian action of L-DOPA. Here, we seek to take advantage of a possible additional D 2 -agonist effect of LY-404,039 and see if an anti-parkinsonian benefit might be achieved in addition to the antidyskinetic effect of mGlu 2/3 activation. To this end, we have administered LY-404,039 (vehicle, 0.1, 1 and 10â mg/kg) to 6-OHDA-lesioned rats, after which the severity of axial, limbs and oro-lingual (ALO) AIMs was assessed. The addition of LY-404,039 10â mg/kg to L-DOPA resulted in a significant reduction of ALO AIMs over 60-100 min (54%, P â <â 0.05). In addition, LY-404,039 significantly enhanced the antiparkinsonian effect of L-DOPA, assessed through the cylinder test (76%, P â <â 0.01). These results provide further evidence that mGlu 2/3 orthosteric stimulation may alleviate dyskinesia in PD and, in the specific case of LY-404,039, a possible D 2 -agonist effect might also make it attractive to address motor fluctuations. Because LY-404,039 and its pro-drug have been administered to humans, they could possibly be advanced to Phase IIa trials rapidly for the treatment of motor complications in PD.
Asunto(s)
Discinesia Inducida por Medicamentos , Trastornos Parkinsonianos , Receptores de Glutamato Metabotrópico , Animales , Masculino , Ratas , Aminoácidos/farmacología , Antiparkinsonianos/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Discinesia Inducida por Medicamentos/tratamiento farmacológico , Agonistas de Aminoácidos Excitadores/farmacología , Levodopa/farmacología , Oxidopamina , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/tratamiento farmacológico , Ratas Sprague-Dawley , Ratas Wistar , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
Polyionic liquid hydrogels attract increasing attention due to their unique properties and potential applications. However, research on amino acid-based polyionic liquid hydrogels is still in its infancy stage. Moreover, the effect of amino acid types on the properties of hydrogels is rarely studied to date. In this work, amino acid-based polyionic liquid hydrogels (D/L-PCAA hydrogels) are synthesized by copolymerizing vinyl choline-amino acid ionic liquids and acrylic acids using Al3+ as a crosslinking agent and bacterial cellulose (BC) as a reinforcing agent. The effects of amino acid types on mechanical and antimicrobial properties are systematically investigated. D-arginine-based hydrogel (D-PCArg) shows the highest tensile strength (220.7 KPa), D-phenylalanine-based hydrogel (D-PCPhe) exhibits the highest elongation at break (1346%), and L-aspartic acid-based hydrogel (L-PCAsp) has the highest elastic modulus (206.9 KPa) and toughness (1.74 MJ m-3). D/L-PCAsp hydrogels demonstrate stronger antibacterial capacity against Escherichia coli and Staphylococcus aureus, and D/L-PCPhe hydrogels possess higher antifungal activity against Cryptococcus neoformans. Moreover, the resultant hydrogels exhibit prominent hemocompatibility and low toxicity, as well as excellent self-healing capabilities (86%) and conductivity (2.8 S m-1). These results indicate that D/L-PCAA hydrogel provides a promise for applications in wound dressings.
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Aminoácidos , Antibacterianos , Escherichia coli , Hidrogeles , Líquidos Iónicos , Staphylococcus aureus , Hidrogeles/química , Hidrogeles/farmacología , Hidrogeles/síntesis química , Escherichia coli/efectos de los fármacos , Aminoácidos/química , Aminoácidos/farmacología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Antibacterianos/farmacología , Antibacterianos/síntesis química , Líquidos Iónicos/química , Líquidos Iónicos/farmacología , Pruebas de Sensibilidad Microbiana , Antifúngicos/farmacología , Antifúngicos/química , Antifúngicos/síntesis química , Antiinfecciosos/química , Antiinfecciosos/farmacología , Antiinfecciosos/síntesis químicaRESUMEN
The Warburg effect links growth and glycolysis in cancer. A key purpose of the Warburg effect is to generate glycolytic intermediates for anabolic reactions, such as nucleotides â RNA/DNA and amino acids â protein synthesis. The aim of this study was to investigate whether a similar 'glycolysis-for-anabolism' metabolic reprogramming also occurs in hypertrophying skeletal muscle. To interrogate this, we first induced C2C12 myotube hypertrophy with IGF-1. We then added 14C glucose to the differentiation medium and measured radioactivity in isolated protein and RNA to establish whether 14C had entered anabolism. We found that especially protein became radioactive, suggesting a glucose â glycolytic intermediates â non-essential amino acid(s) â protein series of reactions, the rate of which was increased by IGF-1. Next, to investigate the importance of glycolytic flux and non-essential amino acid synthesis for myotube hypertrophy, we exposed C2C12 and primary mouse myotubes to the glycolysis inhibitor 2-Deoxy-d-glucose (2DG). We found that inhibiting glycolysis lowered C2C12 and primary myotube size. Similarly, siRNA silencing of PHGDH, the key enzyme of the serine biosynthesis pathway, decreased C2C12 and primary myotube size; whereas retroviral PHGDH overexpression increased C2C12 myotube size. Together these results suggest that glycolysis is important for hypertrophying myotubes, which reprogram their metabolism to facilitate anabolism, similar to cancer cells.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina , Neoplasias , Animales , Ratones , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fosfoglicerato-Deshidrogenasa/genética , Fosfoglicerato-Deshidrogenasa/metabolismo , Fosfoglicerato-Deshidrogenasa/farmacología , Fibras Musculares Esqueléticas/metabolismo , Neoplasias/metabolismo , ARN/metabolismo , Hipertrofia/metabolismo , Glucosa/farmacología , Aminoácidos/genética , Aminoácidos/metabolismo , Aminoácidos/farmacologíaRESUMEN
As a vital hallmarker of cancer, the metabolic reprogramming has been shown to play a pivotal role in tumour occurrence, metastasis and drug resistance. Amongst a vast variety of signalling molecules and metabolic enzymes involved in the regulation of cancer metabolism, two key transcription factors Nrf1 and Nrf2 are required for redox signal transduction and metabolic homeostasis. However, the regulatory effects of Nrf1 and Nrf2 (both encoded by Nfe2l1 and Nfe2l2, respectively) on the metabolic reprogramming of hepatocellular carcinoma cells have been not well understood to date. Here, we found that the genetic deletion of Nrf1 and Nrf2 from HepG2 cells resulted in distinct metabolic reprogramming. Loss of Nrf1α led to enhanced glycolysis, reduced mitochondrial oxygen consumption, enhanced gluconeogenesis and activation of the pentose phosphate pathway in the hepatocellular carcinoma cells. By striking contrast, loss of Nrf2 attenuated the glycolysis and gluconeogenesis pathways, but with not any significant effects on the pentose phosphate pathway. Moreover, knockout of Nrf1α also caused fat deposition and increased amino acid synthesis and transport, especially serine synthesis, whilst Nrf2 deficiency did not cause fat deposition, but attenuated amino acid synthesis and transport. Further experiments revealed that such distinctive metabolic programming of between Nrf1α-/- and Nrf2-/- resulted from substantial activation of the PI3K-AKT-mTOR signalling pathway upon the loss of Nrf1, leading to increased expression of critical genes for the glucose uptake, glycolysis, the pentose phosphate pathway, and the de novo lipid synthesis, whereas deficiency of Nrf2 resulted in the opposite phenomenon by inhibiting the PI3K-AKT-mTOR pathway. Altogether, these provide a novel insight into the cancer metabolic reprogramming and guide the exploration of a new strategy for targeted cancer therapy.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Reprogramación Metabólica , Factor 1 Relacionado con NF-E2 , Factor 2 Relacionado con NF-E2 , Humanos , Aminoácidos/farmacología , Células Hep G2 , Neoplasias Hepáticas/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Factor 1 Relacionado con NF-E2/genética , Factor 1 Relacionado con NF-E2/metabolismoRESUMEN
INTRODUCTION: Peritoneal dialysis (PD) is a life maintaining treatment in patients with end-stage renal disease. Its chronic application leads to peritoneal mesothelial layer denudation and fibrotic transformation along with vascular activation of inflammatory pathways. The impact of different PD fluids (PDF) on mesothelial and endothelial cell function and repair mechanisms are not comprehensively described. MATERIALS AND METHODS: Mesothelial (MeT-5A) and endothelial cells (EA.hy926) were cultured in 1:1 ratio with cell medium and different PDF (icodextrin-based, amino acid-based, and glucose-based). Cell adhesion, cell migration, and cell proliferation in 2D and spheroid formation and collagen gel contraction assays in 3D cell cultures were performed. RESULTS: Cell proliferation and cell-mediated gel contraction were both significantly decreased in all conditions. 3D spheroid formation was significantly reduced with icodextrin and amino acid PDF, but unchanged with glucose PDF. Adhesion was significantly increased by amino acid PDF in mesothelial cells and decreased by icodextrin and amino acid PDF in endothelial cells. Migration capacity was significantly decreased in mesothelial cells by all three PDF, while endothelial cells remained unaffected. CONCLUSIONS: In 3D phenotypes the effects of PDF are more uniform in both mesothelial and endothelial cells, mitigating spheroid formation and gel contraction. On the contrary, effects on 2D phenotypes are more uniform in the icodextrin and amino acid PDF as opposed to glucose ones and affect mesothelial cells more variably. 2D and 3D comparative assessments of PDF effects on the main peritoneal membrane cell barriers, the mesothelial and endothelial, could provide useful translational information for PD studies.
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Células Endoteliales , Diálisis Peritoneal , Humanos , Icodextrina/metabolismo , Icodextrina/farmacología , Soluciones para Diálisis/efectos adversos , Soluciones para Diálisis/metabolismo , Peritoneo/metabolismo , Fenotipo , Aminoácidos/metabolismo , Aminoácidos/farmacología , Glucosa/farmacología , Glucosa/metabolismo , Células Cultivadas , Células EpitelialesRESUMEN
Embryonic transfer of bovine blastocysts produced by in vitro fertilization is widely utilized-despite a compromised conception rate. It has been suggested that a set of four evaluation criteria for judging the quality of embryos, based on the timing of early cleavages and proper morphologies of embryos, can effectively predict pregnancy success. These blastocysts are hereafter referred to as four-criteria-compliant blastocysts. The same criteria should be used to modify the culture media to improve embryo quality. For example, culture media is often supplemented with nonessential amino acids (NEAA) at a uniform concentration despite the major variation in their concentration in the oviductal fluid. In the present study, the effects of the embryo culture medium, namely CR1, supplemented with all seven MEM NEAA or six of them, excluding one at a time, were examined. All media, except for the medium that did not contain proline and serine, tended to improve the efficiency of producing four-criteria-compliant blastocysts, and excluding alanine was particularly effective. The absence of alanine resulted in the rapid occurrence of the first cleavage and pronuclear formation of fertilized oocytes in the alanine-free medium compared to that in the medium containing alanine. These results suggested that alanine hinders certain events involved in the progression of early embryogenesis, which is necessary to achieve the four criteria that provide a benchmark for pregnancy. Therefore, a significantly higher percentage of embryos satisfied the recommended criteria and developed into four-criteria-compliant blastocysts when developed in alanine-free medium than in alanine-containing medium.
Asunto(s)
Alanina , Blastocisto , Medios de Cultivo , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Fertilización In Vitro , Oocitos , Animales , Bovinos , Femenino , Fertilización In Vitro/veterinaria , Fertilización In Vitro/métodos , Alanina/farmacología , Técnicas de Cultivo de Embriones/veterinaria , Técnicas de Cultivo de Embriones/métodos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Blastocisto/efectos de los fármacos , Embarazo , Aminoácidos/farmacología , Aminoácidos/metabolismoRESUMEN
OBJECTIVE: This study aimed to investigate the ultraviolet (UV) protection/repair benefits of a patented Amino Acid Complex (AAComplex). METHODS: I) AAComplex was incubated with dermal fibroblasts, with/without UVA, and collagen I was measured with a GlasBoxPlus device. II) A lotion, with/without AAComplex (1%) was applied topically to skin explants, following UVA irradiation, and quantified for health-related biomarkers (TNFalpha, histamine, and MMP-1). III) A broad spectrum sunscreen with SPF 46 and a skincare serum containing AAComplex (2%) were assessed using epidermal equivalents, in the presence of UV irradiation, for effects on IL-1alpha, thymine dimers, Ki-67, filaggrin and Nrf2. RESULTS: I) Collagen I synthesis in dermal fibroblasts was significantly decreased after UVA compared to without UV. The presence of AAComplex prevented this decrease. II) UVA irradiation of skin explants increased histamine, TNFα, and MMP-1. Hydrocortisone aceponate cream significantly decreases all 3 biomarkers. AAComplex contained lotion also significantly decreased all 3 biomarkers, the no AAComplex control lotion only reduced histamine. III) With the regimen of sunscreen + AAComplex contained skincare serum, the significant reduction in IL-1alpha was observed along with a complete recovery of Ki-67 and stimulation of filaggrin and Nrf2T. No thymine dimer positive cell was observed indicating the most positive skin impact from the regiment. Conclusion: This research using different human skin models demonstrated that AAComplex can provide protection and damage repair caused by UV, at the ingredient level also when formulated in a serum or lotion formula. Skin may be best protected from UV damage when the regimen is used. J Drugs Dermatol. 2024;23(5):366-375. doi:10.36849/JDD.7916.
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Fibroblastos , Proteínas Filagrina , Metaloproteinasa 1 de la Matriz , Factor 2 Relacionado con NF-E2 , Factor de Necrosis Tumoral alfa , Rayos Ultravioleta , Humanos , Rayos Ultravioleta/efectos adversos , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Fibroblastos/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Piel/efectos de la radiación , Piel/efectos de los fármacos , Piel/metabolismo , Protectores Solares/administración & dosificación , Protectores Solares/química , Protectores Solares/farmacología , Aminoácidos/administración & dosificación , Aminoácidos/farmacología , Aminoácidos/química , Interleucina-1alfa/metabolismo , Histamina/sangre , Crema para la Piel/administración & dosificación , Biomarcadores/metabolismo , Colágeno Tipo I , Proteínas de Filamentos Intermediarios/metabolismo , Antígeno Ki-67/metabolismo , Dímeros de Pirimidina , Células CultivadasRESUMEN
In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.
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Factor de Transcripción Activador 4/genética , Edición Génica/métodos , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mitocondrias/genética , Proteínas Serina-Treonina Quinasas/genética , 3-Hidroxiacil-CoA Deshidrogenasas/genética , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Factor de Transcripción Activador 4/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aminoácidos/deficiencia , Aminoácidos/farmacología , Antimicina A/análogos & derivados , Antimicina A/farmacología , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Medios de Cultivo/química , Medios de Cultivo/farmacología , Regulación de la Expresión Génica , Genoma Humano , Glucosa/deficiencia , Glucosa/farmacología , Células HEK293 , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oligomicinas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , Transducción de Señal , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Antimicrobial peptides (AMPs) are promising therapeutic agents against bacteria. We have previously reported an amphipathic AMP Stripe composed of cationic L-Lys and hydrophobic L-Leu/L-Ala residues, and Stripe exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria. Gramicidin A (GA), composed of repeating sequences of L- and D-amino acids, has a unique ß6.3-helix structure and exhibits broad antimicrobial activity. Inspired by the structural properties and antimicrobial activities of LD-alternating peptides such as GA, in this study, we designed Stripe derivatives with LD-alternating sequences. We found that simply alternating L- and D-amino acids in the Stripe sequence to give StripeLD caused a reduction in antimicrobial activity. In contrast, AltStripeLD, with cationic and hydrophobic amino acids rearranged to yield an amphipathic distribution when the peptide adopts a ß6.3-helix, displayed higher antimicrobial activity than AltStripe. These results suggest that alternating L-/D-cationic and L-/D-hydrophobic amino acids in accordance with the helical structure of an AMP may be a useful way to improve antimicrobial activity and develop new AMP drugs.
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Aminoácidos , Antibacterianos , Aminoácidos/farmacología , Antibacterianos/química , Péptidos Antimicrobianos , Bacterias Gramnegativas , Relación Estructura-Actividad , Bacterias Grampositivas , Estructura Secundaria de Proteína , Gramicidina/química , Péptidos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
BACKGROUND: Cervical cancer, a life-threatening disease, is the seventh most commonly detected cancer among women throughout the world. The present study investigated the effect of tretinoin on cervical cancer growth and metastasis in vitro and in vivo in the mice model. MATERIALS AND METHODS: Cell Counting Kit-8, clonogenic survival, and transwell chamber assays were used for determination cells proliferation, colony formation, and invasiveness. Western blotting assay was used for assessment of protein expression whereas AutoDock Vina and Discovery studio software for in silico studies. RESULTS: Tretinoin treatment significantly (p < .05) reduced the proliferation of HT-3 and Caski cells in concentration-based manner. Incubation with tretinoin caused a significant decrease in clonogenic survival of HT-3 and Caski cells compared with the control cultures. The invasive potential of HT-3 cells was decreased to 18%, whereas that of Caski cells to 21% on treatment with 8 µM concentration of tretinoin. In HT-3 cells, tretinoin treatment led to a prominent reduction in p-focal adhesion kinase (FAK), matrix metalloproteinases (MMP)-2, and MMP-9 expression in HT-3 cells. Treatment of the cervical cancer mice model with tretinoin led to a prominent decrease in tumor growth. The metastasis of tumor in model cervical cancer mice group was effectively inhibited in spleen, intestines, and peritoneal cavity. In silico studies showed that tretinoin interacts with alanine, proline, isoleucine, and glycine amino acid residues of FAK protein to block its activation. The 2-dimensional diagram of interaction of tretinoin with FAK protein revealed that tretinoin binds to alanine and glycine amino acids through conventional hydrogen bonding. CONCLUSION: In summary, tretinoin suppressed the proliferation, colony formation, and invasiveness of cervical cancer cells in vitro. It decreased the expression of activated focal adhesion kinase, MMP-2, and MMP-9 in HT-3 cells in dose-dependent manner. In silico studies showed that tretinoin interacts with alanine and glycine amino acids through conventional hydrogen bonding. In vivo data demonstrated that treatment of the cervical cancer mice model with tretinoin led to a prominent decrease in tumor growth. Therefore, tretinoin can be developed as an effective therapeutic agent for cervical cancer treatment.
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Neoplasias del Cuello Uterino , Humanos , Femenino , Animales , Ratones , Neoplasias del Cuello Uterino/metabolismo , Tretinoina/farmacología , Tretinoina/uso terapéutico , Línea Celular Tumoral , Regulación hacia Abajo , Metaloproteinasa 9 de la Matriz/metabolismo , Proliferación Celular , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Alanina/metabolismo , Alanina/farmacología , Alanina/uso terapéutico , Glicina/metabolismo , Glicina/farmacología , Glicina/uso terapéutico , Aminoácidos/metabolismo , Aminoácidos/farmacología , Aminoácidos/uso terapéutico , Invasividad Neoplásica , Movimiento CelularRESUMEN
Some new hemorphin-4 analogs with structures of Xxx-Pro-Trp-Thr-NH2 and Tyr-Yyy-Trp-Thr-NH2, where Xxx is 2-amino-3-(4-hydroxy-2,6-dimethylphenyl)propanoic acid or 2-amino-3-(4-dibenzylamino-2,6-dimethylphenyl)propanoic acid, and Yyy is (2S,4S)-4-amino-pyrrolidine-2-carboxylic acid, were synthesized and characterized by electrochemical and spectral analyses. In vivo anticonvulsant and antinociceptive activities of peptide derivatives were studied after intracerebroventricular injection in mice. The therapeutic effects of the modified peptides on seizures and pain in mice were evaluated to provide valuable insights into the potential applications of the novel compounds. Electrochemical characterization showed that the compounds behave as weak protolytes and that they are in a soluble, stable molecular form at physiological pH values. The antioxidant activity of the peptides was evaluated with voltammetric analyses, which were confirmed by applying the 2,2-Diphenyl-1-picrylhydrazyl method. The compounds showed satisfactory results regarding their structural stability, reaching the desired centers for the manifestation of biological activity without hydrolysis processes at 37°C and physiological pH. Dm-H4 and H4-P1 exhibited 100% and 83% potency to suppress the psychomotor seizures in the 6-Hz test compared to 67% activity of H4. Notably, only the H4-P1 had efficacy in blocking the tonic component in the maximal electroshock test with a potency comparable to H4. All investigated peptides containing unnatural conformationally restricted amino acids showed antinociceptive effects. The analogs Db-H4 and H4-P1 showed the most pronounced and long-lasting effect in both experimental models of pain induced by thermal and chemical stimuli. Dm-H4 produced a dose-dependent thermal antinociception and H4-P2 inhibited only formalin-induced pain behavior.
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Convulsiones , Animales , Ratones , Masculino , Convulsiones/tratamiento farmacológico , Relación Estructura-Actividad , Anticonvulsivantes/farmacología , Anticonvulsivantes/síntesis química , Anticonvulsivantes/química , Dolor/tratamiento farmacológico , Aminoácidos/química , Aminoácidos/farmacología , Aminoácidos/síntesis química , Antioxidantes/farmacología , Antioxidantes/síntesis química , Antioxidantes/química , Analgésicos/farmacología , Analgésicos/síntesis química , Analgésicos/química , Relación Dosis-Respuesta a Droga , Modelos Animales de Enfermedad , Péptidos Opioides/farmacología , Péptidos Opioides/síntesis química , Péptidos Opioides/química , Analgésicos Opioides/farmacología , Analgésicos Opioides/síntesis química , Analgésicos Opioides/químicaRESUMEN
OBJECTIVES: An adjunct in non-surgical periodontal therapy might be sodium hypochlorite (NaOCl)-based agents. The purpose of the present in vitro study was to get deeper knowledge on the influence of different parameters as time after mixing, pH, and chemical composition of an amino acid 0.475% NaOCl (AA-NaOCl) gel consisting of two components on its anti-biofilm activity. MATERIALS AND METHODS: Six-species biofilms were cultured for 5 days, before AA-NaOCl gel was applied. In the different series, the influence of the time after mixing of the two components before application, of the concentration of NaOCl in the gel mixture, of the pH of the gel mixture, and of an exchange of the amino acid component by hyaluronic acid (HA), was analyzed. RESULTS: Mixing time point experiments showed that the AA-NaOCl gel is capable of statistically significantly reducing colony-forming unit (cfu) counts up to 30 min after mixing, but only up to 20 min after mixing the reduction was more than 2 log10 cfu. The pH experiments indicate that a reduced pH results in a reduced activity of the NaOCl formulation. NaOCl concentrations in the formulation in the range from 0.475 to 0.2% provide adequate activity on biofilms. A HA/NaOCl gel was equally active against the biofilm as the AA-NaOCl gel. CONCLUSION: Mixing of the components should be made in a timeframe of 20 min before applications. An optimization of the composition of the NaOCl formulation might be possible and should be a topic in further in vitro studies. CLINICAL RELEVANCE: The AA-NaOCl gel formulation can be mixed up to 20 min before application. Further, the study indicates that the composition of the NaOCl gel formulation can be optimized.
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Enfermedades Periodontales , Hipoclorito de Sodio , Humanos , Hipoclorito de Sodio/farmacología , Hipoclorito de Sodio/química , Enterococcus faecalis , Enfermedades Periodontales/tratamiento farmacológico , Bacterias , Aminoácidos/farmacologíaRESUMEN
In this study, a series of novel benzyloxybenzene substituted (S)-α-amino acid methyl esters and their amide derivatives were synthesized and evaluated for their inhibitory actions against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), monoamine oxidase A (MAO-A), and monoamine oxidase B (MAO-B). The synthetic strategy was based on starting from benzyl bromide (5) and 4-hydroxybenzaldehyde (6). The reaction of 5 and 6 in the presence of K2 CO3 gave benzyloxybenzaldehyde 7. Benzyloxybenzene substituted (S)-α-amino acid methyl esters 11, 12, 13, (±)-19, and (±)-20 were obtained from the reaction of L-amino acid methyl esters with benzyloxybenzaldehyde (7) followed by in situ reduction with NaBH4 . The reaction of (S)-11, (S)-12, 13, (±)-19, and (±)-20 with excess ammonia gave amides (S)-14, (S)-15, 16, (±)-21, and (±)-22. The in vitro inhibitory activities of compounds against MAO-A, MAO-B, AChE, and BChE were investigated. Within the α-amino acid methyl ester series, 13 (21.32 ± 0.338 µM) showed selectivity by inhibiting the MAO-B better than MAO-A. 13 emerged as the most active member of this series, exhibiting a 12-fold selectivity for MAO-B. 14 (4.501 ± 0.295 µM) demonstrated a pronounced selectivity for MAO-A over MAO-B, with a selectivity ratio of 110-fold. In addition, it was determined that compound 15 (95.65 ± 3.09 µM) had high selectivity for BChE inhibition. 21 was demonstrated the most potent inhibition (18.36 ± 1.36 µM) against AChE.
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Acetilcolinesterasa , Butirilcolinesterasa , Amidas/farmacología , Aminoácidos/farmacología , Ésteres , MonoaminooxidasaRESUMEN
Skeletal muscle atrophy (SMA) is caused by a rise in muscle breakdown and a decline in protein synthesis, with a consequent loss of mass and function. This study characterized the effect of an amino acid mixture (AA) in models of SMA, focusing on mitochondria. C57/Bl6 mice underwent immobilization of one hindlimb (I) or cardiotoxin-induced muscle injury (C) and were compared with controls (CTRL). Mice were then administered AA in drinking water for 10 days and compared to a placebo group. With respect to CTRL, I and C reduced running time and distance, along with grip strength; however, the reduction was prevented by AA. Tibialis anterior (TA) muscles were used for histology and mitochondria isolation. I and C resulted in TA atrophy, characterized by a reduction in both wet weight and TA/body weight ratio and smaller myofibers than those of CTRL. Interestingly, these alterations were lightly observed in mice treated with AA. The mitochondrial yield from the TA of I and C mice was lower than that of CTRL but not in AA-treated mice. AA also preserved mitochondrial bioenergetics in TA muscle from I and C mice. To conclude, this study demonstrates that AA prevents loss of muscle mass and function in SMA by protecting mitochondria.
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Aminoácidos , Metabolismo Energético , Ratones Endogámicos C57BL , Músculo Esquelético , Atrofia Muscular , Animales , Ratones , Metabolismo Energético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Aminoácidos/farmacología , Aminoácidos/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/patología , Atrofia Muscular/etiología , Masculino , Modelos Animales de Enfermedad , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/patología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacosRESUMEN
Isoliquiritigenin (ISL) is a chalcone that has shown great potential in the treatment of cancer. However, its relatively weak activity and low water solubility limit its clinical application. In this study, we designed and synthesized 21 amino acid ester derivatives of ISL and characterized the compounds using 1H NMR and 13C NMR. Among them, compound 9 (IC50 = 14.36 µM) had a better inhibitory effect on human cervical cancer (Hela) than ISL (IC50 = 126.5 µM), and it was superior to the positive drug 5-FU (IC50 = 33.59 µM). The mechanism of the action experiment showed that compound 9 could induce Hela cell apoptosis and autophagy through the PI3K/Akt/mTOR pathway.