RESUMEN
The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern1. For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings2. Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the [Formula: see text] nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Antibacterianos/química , Antibacterianos/clasificación , Clindamicina/síntesis química , Clindamicina/farmacología , Descubrimiento de Drogas , Lincomicina/síntesis química , Lincomicina/farmacología , Metiltransferasas/genética , Metiltransferasas/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Oxepinas , Piranos , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/química , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Thermus thermophilus/efectos de los fármacos , Thermus thermophilus/enzimología , Thermus thermophilus/genéticaRESUMEN
The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Antibacterianos/química , Farmacorresistencia Bacteriana/efectos de los fármacos , Ribosomas/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , ARN Ribosómico 23S/química , ARN Ribosómico 23S/metabolismo , Metiltransferasas/metabolismo , Metiltransferasas/química , Metiltransferasas/antagonistas & inhibidores , Metilación , Modelos Moleculares , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/genéticaRESUMEN
Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Genes Bacterianos , Bacterias Grampositivas , Antibacterianos/farmacología , Antibacterianos/química , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/genética , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/genética , Nucleósidos/química , Nucleósidos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Genes Bacterianos/genética , Clostridium/efectos de los fármacos , Clostridium/genética , Microscopía por CrioelectrónRESUMEN
BACKGROUND AND AIMS: Metabolism in the liver is dysregulated in obesity, contributing to various health problems including steatosis and insulin resistance. While the pathogenesis of lipid accumulation has been extensively studied, the protective mechanism against lipid challenge in the liver remains unclear. Here, we report that Src homology 3 domain binding kinase 1 (SBK1) is a regulator of hepatic lipid metabolism and systemic insulin sensitivity in response to obesity. APPROACH AND RESULTS: Enhanced Sbk1 expression was found in the liver of high-fat diet (HFD)-induced obese mice and fatty acid (FA)-challenged hepatocytes. SBK1 knockdown in mouse liver cells augmented FA uptake and lipid accumulation. Similarly, liver-specific SBK1 knockout ( Lsko ) mice displayed more severe hepatosteatosis and higher expression of genes in FA uptake and lipogenesis than the Flox/Flox ( Fl/Fl ) control mice when fed the HFD. The HFD-fed Lsko mice also showed symptoms of hyperglycemia, poor systemic glucose tolerance, and lower insulin sensitivity than the Fl/Fl mice. On the other hand, hepatic Sbk1 overexpression alleviated the high-fructose diet-induced hepatosteatosis, hyperlipidemia, and hyperglycemia in mice. White adipose tissue browning was also observed in hepatic SBK1 -overexpressed mice. Moreover, we found that SBK1 was a positive regulator of FGF21 in the liver during energy surplus conditions. Mechanistically, SBK1 phosphorylates the orphan nuclear receptor 4A1 (Nur77) on serine 344 to promote hepatic FGF21 expression and inhibit the transcription of genes involved in lipid anabolism. CONCLUSIONS: Collectively, our data suggest that SBK1 is a regulator of the metabolic adaption against obesity through the Nur77-FGF21 pathway.
Asunto(s)
Hígado Graso , Resistencia a la Insulina , Proteínas Quinasas , Animales , Ratones , Dieta Alta en Grasa/efectos adversos , Hígado Graso/metabolismo , Hígado Graso/patología , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Hiperglucemia/patología , Lípidos , Hígado/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/complicaciones , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores NuclearesRESUMEN
Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression.
Asunto(s)
Antibacterianos , Bacillus subtilis , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Farmacorresistencia Microbiana/genética , Regulación Bacteriana de la Expresión Génica , Guanosina Pentafosfato/metabolismo , Factores R , Transcripción GenéticaRESUMEN
The regioselectivity of a [3+2] dipolar cycloaddition reaction of a stabilized azomethine ylide with an electron-deficient dipolarophile was found to be counter to a report published in this journal.
RESUMEN
Hydrogen-tritium exchange is widely employed for radioisotopic labeling of molecules of biological interest but typically involves the metal-promoted exchange of sp2-hybridized carbon-hydrogen bonds, a strategy that is not directly applicable to the antibiotic iboxamycin, which possesses no such bonds. We show that ruthenium-induced 2'-epimerization of 2'-epi-iboxamycin in HTO (200 mCi) of low specific activity (10 Ci/g, 180 mCi/mmol) at 80 °C for 18 h affords after purification tritium-labeled iboxamycin (3.55 µCi) with a specific activity of 53 mCi/mmol. Iboxamycin displayed an apparent inhibition constant (Ki, app) of 41 ± 30 nM towards Escherichia coli ribosomes, binding approximately 70-fold more tightly than the antibiotic clindamycin (Ki, app = 2.7 ± 1.1 µM).
Asunto(s)
Antibacterianos , Clindamicina , Antibacterianos/química , Clindamicina/química , Clindamicina/metabolismo , Hidrógeno , Tritio/química , Rutenio/químicaRESUMEN
N-Nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N-nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product l-alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the source of the N-nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non-proteinogenic amino acid l-2,3-diaminopropionic acid (l-Dap) is synthesized and loaded onto a free-standing peptidyl carrier protein (PCP) domain in l-alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated enâ route to the diazeniumdiolate. These discoveries will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.
Asunto(s)
Alanina/análogos & derivados , Compuestos Azo/metabolismo , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Familia de Multigenes , Streptomyces/metabolismo , Alanina/metabolismo , Proteínas Bacterianas/genética , Estructura Molecular , Streptomyces/genéticaRESUMEN
Adipose tissue as the largest energy reservoir and endocrine organ is essential for maintenance of systemic glucose, lipid and energy homeostasis, but these metabolic functions decline with ageing and obesity. Adipose tissue senescence is one of the common features in obesity and ageing. Although cellular senescence is a defensive mechanism preventing tumorigenesis, its occurrence in adipose tissue causatively induces defective adipogenesis, inflammation, aberrant adipocytokines production and insulin resistance, leading to adipose tissue dysfunction. In addition to these paracrine effects, adipose tissue senescence also triggers systemic inflammation and senescence as well as insulin resistance in the distal metabolic organs, resulting in Type 2 diabetes and other premature physiological declines. Multiple cell types including mature adipocytes, immune cells, endothelial cells and progenitor cells gradually senesce at different levels in different fat depots with ageing and obesity, highlighting the heterogeneity and complexity of adipose tissue senescence. In this review, we discuss the causes and consequences of adipose tissue senescence, and the major cell types responsible for adipose tissue senescence in ageing and obesity. In addition, we summarize the pharmacological approaches and lifestyle intervention targeting adipose tissue senescence for the treatment of obesity- and ageing-related metabolic diseases.
Asunto(s)
Tejido Adiposo/patología , Envejecimiento/patología , Enfermedades Metabólicas/patología , Obesidad/patología , Animales , Humanos , Terapia Molecular Dirigida , Transducción de SeñalRESUMEN
Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat-insulin-promoter-Cre (RIP-Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT Genetic ablation of APPL2 in RIP-Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP-Cre neurons, inactivation of VMH AMPK, or treatment with a ß3-adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP-Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP-Cre neurons, in which the APPL2-AMPK signaling axis is crucial for this defending mechanism to cold and obesity.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Neuronas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Sistema Nervioso Simpático/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Tejido Adiposo Beige/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Metabolismo Energético , Eliminación de Gen , Técnicas de Sustitución del Gen , Genotipo , Hipotálamo/metabolismo , Ratones , Ratones Noqueados , Fenotipo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , TermogénesisRESUMEN
Adipose tissue is an active endocrine and immune organ that controls systemic immunometabolism via multiple pathways. Diverse immune cell populations reside in adipose tissue, and their composition and immune responses vary with nutritional and environmental conditions. Adipose tissue dysfunction, characterized by sterile low-grade chronic inflammation and excessive immune cell infiltration, is a hallmark of obesity, as well as an important link to cardiometabolic diseases. Amongst the pro-inflammatory factors secreted by the dysfunctional adipose tissue, interleukin (IL)-1ß, induced by the NLR family pyrin domain-containing 3 (NLRP3) inflammasome, not only impairs peripheral insulin sensitivity, but it also interferes with the endocrine and immune functions of adipose tissue in a paracrine manner. Human studies indicated that NLRP3 activity in adipose tissues positively correlates with obesity and its metabolic complications, and treatment with the IL-1ß antibody improves glycaemia control in type 2 diabetic patients. In mouse models, genetic or pharmacological inhibition of NLRP3 activation pathways or IL-1ß prevents adipose tissue dysfunction, including inflammation, fibrosis, defective lipid handling and adipogenesis, which in turn alleviates obesity and its related metabolic disorders. In this review, we summarize both the negative and positive regulators of NLRP3 inflammasome activation, and its pathophysiological consequences on immunometabolism. We also discuss the potential therapeutic approaches to targeting adipose tissue inflammasome for the treatment of obesity and its related metabolic disorders.
Asunto(s)
Tejido Adiposo/metabolismo , Inflamasomas/metabolismo , Enfermedades Metabólicas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Adipoquinas/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/patología , Animales , Autofagia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Humanos , Resistencia a la Insulina , Metabolismo de los Lípidos , Lipopolisacáridos/farmacología , Enfermedades Metabólicas/tratamiento farmacológico , Enfermedades Metabólicas/patología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Obesidad/metabolismo , Obesidad/patología , Paniculitis/metabolismo , Paniculitis/patologíaRESUMEN
AIMS/HYPOTHESIS: Beta cell inflammation and demise is a feature of type 1 diabetes. The insulin-sensitising molecule 'adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1' (APPL1), which contains an NH2-terminal Bin/Amphiphysin/Rvs domain, a central pleckstrin homology domain and a COOH-terminal phosphotyrosine-binding domain, has been shown to modulate inflammatory response in various cell types but its role in regulating beta cell mass and inflammation in type 1 diabetes remains unknown. Thus, we investigated whether APPL1 prevents beta cell apoptosis and inflammation in diabetes. METHODS: Appl1-knockout mice and their wild-type littermates, as well as C57BL/6N mice injected with adeno-associated virus encoding APPL1 or green fluorescent protein, were treated with multiple-low-dose streptozotocin (MLDS) to induce experimental type 1 diabetes. Their glucose metabolism and beta cell function were assessed. The effect of APPL1 deficiency on beta cell function upon exposure to a diabetogenic cytokine cocktail (CKS; consisting of TNF-α, IL-1ß and IFN-γ) was assessed ex vivo. RESULTS: Expression of APPL1 was significantly reduced in pancreatic islets from mouse models of type 1 diabetes or islets treated with CKS. Hyperglycaemia, beta cell loss and insulitis induced by MLDS were exacerbated by genetic deletion of Appl1 but were alleviated by beta cell-specific overexpression of APPL1. APPL1 preserved beta cell mass by reducing beta cell apoptosis upon treatment with MLDS. Mechanistically, APPL1 deficiency potentiate CKS-induced phosphorylation of NFκB inhibitor, α (IκBα) and subsequent phosphorylation and transcriptional activation of p65, leading to a dramatic induction of NFκB-regulated apoptotic and proinflammatory programs in beta cells. Pharmacological inhibition of NFκB or inducible NO synthase (iNOS) largely abrogate the detrimental effects of APPL1 deficiency on beta cell functions. CONCLUSIONS/INTERPRETATION: APPL1 negatively regulates inflammation and apoptosis in pancreatic beta cells by dampening the NFκB-iNOS-NO axis, representing a promising target for treating type 1 diabetes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Inflamación/metabolismo , FN-kappa B/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis/genética , Apoptosis/fisiología , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Diabetes Mellitus Tipo 1/genética , Células HEK293 , Humanos , Inmunohistoquímica , Inflamación/genética , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxidos de Nitrógeno/metabolismo , Páncreas/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Transducción de Señal/fisiología , Estreptozocina/toxicidadRESUMEN
We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes evolutionarily diverse forms of antimicrobial resistance that render modern antibiotics ineffective. CRM exhibits in vitro and in vivo efficacy against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We show that CRM is highly preorganized for ribosomal binding by determining its density functional theory-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Lastly, we report two additional x-ray crystal structures of CRM in complex with bacterial ribosomes separately modified by the ribosomal RNA methylases, chloramphenicol-florfenicol resistance (Cfr) and erythromycin-resistance ribosomal RNA methylase (Erm), revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.
Asunto(s)
Antibacterianos , Hidrocarburos Aromáticos con Puentes , Farmacorresistencia Bacteriana Múltiple , Lincosamidas , Oxepinas , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Eritromicina/química , Eritromicina/farmacología , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Hidrocarburos Aromáticos con Puentes/síntesis química , Hidrocarburos Aromáticos con Puentes/química , Hidrocarburos Aromáticos con Puentes/farmacología , Oxepinas/síntesis química , Oxepinas/química , Oxepinas/farmacología , Lincosamidas/síntesis química , Lincosamidas/química , Lincosamidas/farmacología , Animales , Ratones , Diseño de Fármacos , Ribosomas/químicaRESUMEN
We have developed a pen and writing tablet for use by subjects during fMRI scanning. The pen consists of two jacketed, multi-mode optical fibers routed to the tip of a hollowed-out ball-point pen. The pen has been further modified by addition of a plastic plate to maintain a perpendicular pen-tablet orientation. The tablet is simply a non-metallic frame holding a paper print of continuously varying color gradients. The optical fibers are routed out of the MRI bore to a light-tight box in an adjacent control room. Within the box, light from a high intensity LED is coupled into one of the fibers, while the other fiber abuts a color sensor. Light from the LED exits the pen tip, illuminating a small spot on the tablet, and the resulting reflected light is routed to the color sensor. Given a lookup table of position for each color on the tablet, the coordinates of the pen on the tablet may be displayed and digitized in real-time. While simple and inexpensive, the system achieves sufficient resolution to grade writing tasks testing dysgraphic and dyslexic phenomena.
Asunto(s)
Agrafia/diagnóstico , Agrafia/fisiopatología , Dislexia/diagnóstico , Dislexia/fisiopatología , Imagen por Resonancia Magnética/economía , Imagen por Resonancia Magnética/instrumentación , Interfaz Usuario-Computador , Color , Costos y Análisis de Costo , Humanos , Movimiento (Física) , Factores de TiempoRESUMEN
The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.
RESUMEN
NLRP3 (NLR family pyrin domain containing 3) inflammasome is a potent mediator of inflammation due to its ability to produce the pro-inflammatory cytokines IL1B (interleukin 1 beta) and IL18 in response to numerous danger signals and pathogens. Mitophagy, a selective form of autophagy, restricts NLRP3 inflammasome activation by limiting the mitochondrial-derived danger signals. Here, we demonstrated that the adaptor protein APPL1 together with its interaction partner RAB5 in early endosomes negatively regulate NLRP3 inflammasome activation via induction of mitophagy in macrophages. Hematopoietic-deletion of Appl1 exacerbates systemic NLRP3 inflammasome activation in rodent models under obese or septic conditions. Our study identified a new regulatory network between early endosomes and mitochondria in control of NLRP3 inflammasome activation.
Asunto(s)
Inflamasomas , Mitofagia , Autofagia , Endosomas/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Background: Listeriosis is a food-borne disease caused by the Gram-positive Bacillota (Firmicute) bacterium Listeria monocytogenes. Clinical L. monocytogenes isolates are often resistant to clinically used lincosamide clindamycin, thus excluding clindamycin as a viable treatment option. Objectives: We have established newly developed lincosamide iboxamycin as a potential novel antilisterial agent. Methods: We determined MICs of the lincosamides lincomycin, clindamycin and iboxamycin for L. monocytogenes, Enterococcus faecalis and Bacillus subtilis strains expressing synergetic antibiotic resistance determinants: ABCF ATPases that directly displace antibiotics from the ribosome and Cfr, a 23S rRNA methyltransferase that compromises antibiotic binding. For L. monocytogenes strains, either expressing VgaL/Lmo0919 or lacking the resistance factor, we performed time-kill kinetics and post-antibiotic effect assays. Results: We show that the synthetic lincosamide iboxamycin is highly active against L. monocytogenes and can overcome the intrinsic lincosamide resistance mediated by VgaL/Lmo0919 ABCF ATPase. While iboxamycin is not bactericidal against L. monocytogenes, it displays a pronounced post-antibiotic effect, which is a valuable pharmacokinetic feature. We demonstrate that VmlR ABCF of B. subtilis grants significant (33-fold increase in MIC) protection from iboxamycin, while LsaA ABCF of E. faecalis grants an 8-fold protective effect. Furthermore, the VmlR-mediated iboxamycin resistance is cooperative with that mediated by the Cfr, resulting in up to a 512-fold increase in MIC. Conclusions: While iboxamycin is a promising new antilisterial agent, our findings suggest that emergence and spread of ABCF ARE variants capable of defeating next-generation lincosamides in the clinic is possible and should be closely monitored.
RESUMEN
The emergence of multidrug-resistant strains and hyper-virulent strains of Mycobacterium tuberculosis are big therapeutic challenges for tuberculosis (TB) control. Repurposing bioactive small-molecule compounds has recently become a new therapeutic approach against TB. This study aimed to identify novel anti-TB agents from a library of small-molecule compounds via a rapid screening system. A total of 320 small-molecule compounds were used to screen for their ability to suppress the expression of a key virulence gene, phop, of the M. tuberculosis complex using luminescence (lux)-based promoter-reporter platforms. The minimum inhibitory and bactericidal concentrations on drug-resistant M. tuberculosis and cytotoxicity to human macrophages were determined. RNA sequencing (RNA-seq) was conducted to determine the drug mechanisms of the selected compounds as novel antibiotics or anti-virulent agents against the M. tuberculosis complex. The results showed that six compounds displayed bactericidal activity against M. bovis BCG, of which Ebselen demonstrated the lowest cytotoxicity to macrophages and was considered as a potential antibiotic for TB. Another ten compounds did not inhibit the in vitro growth of the M. tuberculosis complex and six of them downregulated the expression of phoP/R significantly. Of these, ST-193 and ST-193 (hydrochloride) showed low cytotoxicity and were suggested to be potential anti-virulence agents for M. tuberculosis.
RESUMEN
Dysfunctional triglyceride-very low-density lipoprotein (TG-VLDL) metabolism is linked to metabolic-associated fatty liver disease (MAFLD); however, the underlying cause remains unclear. The study shows that hepatic E3 ubiquitin ligase murine double minute 2 (MDM2) controls MAFLD by blocking TG-VLDL secretion. A remarkable upregulation of MDM2 is observed in the livers of human and mouse models with different levels of severity of MAFLD. Hepatocyte-specific deletion of MDM2 protects against high-fat high-cholesterol diet-induced hepatic steatosis and inflammation, accompanied by a significant elevation in TG-VLDL secretion. As an E3 ubiquitin ligase, MDM2 targets apolipoprotein B (ApoB) for proteasomal degradation through direct protein-protein interaction, which leads to reduced TG-VLDL secretion in hepatocytes. Pharmacological blockage of the MDM2-ApoB interaction alleviates dietary-induced hepatic steatohepatitis and fibrosis by inducing hepatic ApoB expression and subsequent TG-VLDL secretion. The effect of MDM2 on VLDL metabolism is p53-independent. Collectively, these findings suggest that MDM2 acts as a negative regulator of hepatic ApoB levels and TG-VLDL secretion in MAFLD. Inhibition of the MDM2-ApoB interaction may represent a potential therapeutic approach for MAFLD treatment.
Asunto(s)
Apolipoproteínas B , Hígado Graso , Lipoproteínas VLDL , Hígado , Obesidad , Proteínas Proto-Oncogénicas c-mdm2 , Triglicéridos , Animales , Apolipoproteínas B/metabolismo , Hígado Graso/etiología , Hígado Graso/metabolismo , Humanos , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Ratones , Obesidad/complicaciones , Proteolisis , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Triglicéridos/metabolismoRESUMEN
Although mitophagy is known to restrict NLRP3 inflammasome activation, the underlying regulatory mechanism remains poorly characterized. Here we describe a type of early endosome-dependent mitophagy that limits NLRP3 inflammasome activation. Deletion of the endosomal adaptor protein APPL1 impairs mitophagy, leading to accumulation of damaged mitochondria producing reactive oxygen species (ROS) and oxidized cytosolic mitochondrial DNA, which in turn trigger NLRP3 inflammasome overactivation in macrophages. NLRP3 agonist causes APPL1 to translocate from early endosomes to mitochondria, where it interacts with Rab5 to facilitate endosomal-mediated mitophagy. Mice deficient for APPL1 specifically in hematopoietic cell are more sensitive to endotoxin-induced sepsis, obesity-induced inflammation and glucose dysregulation. These are associated with increased expression of systemic interleukin-1ß, a major product of NLRP3 inflammasome activation. Our findings indicate that the early endosomal machinery is essential to repress NLRP3 inflammasome hyperactivation by promoting mitophagy in macrophages.