RESUMEN
Ribosome biogenesis is a highly regulated multistep process aided by energy-consuming auxiliary factors. GTPases form the largest class of auxiliary factors used by bacterial, cytosolic, and mitochondrial ribosomes for their maturation. Mtg3, a circularly permuted YqeH family of GTPase, is implicated in the mitoribosome small subunit biogenesis. However, its precise mechanistic role has yet to be characterized. Mtg3 is likely to bind precursor mitoribosome molecules during subunit maturation in vivo. However, this interaction has yet to be observed with mitoribosomes biochemically. In this study, we delineate the specific conditions necessary for preserving the association of Mtg3 with mitoribosomes on a sucrose density gradient. We show that the C-terminal domain of Mtg3 is required for robust binding to the mitoribosome. Furthermore, point mutants likely to abrogate GTP/GDP binding and GTPase activity compromise protein function in vivo. Surprisingly, the association with the mitoribosome was not compromised in mutants likely to be deficient for nucleotide binding/hydrolysis. Thus, our finding supports a model wherein Mtg3 binds to a precursor mitoribosome through its C-terminus to facilitate a conformational change or validate a folding intermediate driven by the GTP/GDP binding and hydrolysis cycle.
RESUMEN
The standard of care for the treatment of chronic hepatitis B (CHB) is typically lifelong treatment with nucleos(t)ide analogs (NAs), which suppress viral replication and provide long-term clinical benefits. However, infectious virus can still be detected in patients who are virally suppressed on NA therapy, which may contribute to the failure of these agents to cure most CHB patients. Accordingly, new antiviral treatment options are being developed to enhance the suppression of hepatitis B virus (HBV) replication in combination with NAs ("antiviral intensification"). Here, we describe GS-SBA-1, a capsid assembly modulator (CAM) belonging to class CAM-E, that demonstrates potent inhibition of extracellular HBV DNA in vitro (EC50 [50% effective concentration] = 19 nM) in HBV-infected primary human hepatocytes (PHHs) as well as in vivo in an HBV-infected immunodeficient mouse model. GS-SBA-1 has comparable activities across HBV genotypes and nucleos(t)ide-resistant mutants in HBV-infected PHHs. In addition, GS-SBA-1 demonstrated in vitro additivity in combination with tenofovir alafenamide (TAF). The administration of GS-SBA-1 to PHHs at the time of infection prevents covalently closed circular DNA (cccDNA) formation and, hence, decreases HBV RNA and antigen levels (EC50 = 80 to 200 nM). Furthermore, GS-SBA-1 prevents the production of extracellular HBV RNA-containing viral particles in vitro. Collectively, these data demonstrate that GS-SBA-1 is a potent CAM that has the potential to enhance viral suppression in combination with an NA.
Asunto(s)
Hepatitis B Crónica , Hepatitis B , Animales , Ratones , Humanos , Hepatitis B Crónica/tratamiento farmacológico , Cápside , Virus de la Hepatitis B , Antivirales/farmacología , Antivirales/uso terapéutico , Proteínas de la Cápside/genética , ARN , ADN Viral/genética , ADN Circular , Hepatitis B/tratamiento farmacológicoRESUMEN
The synthesis of Cox1, the conserved catalytic-core subunit of Complex IV, a multisubunit machinery of the mitochondrial oxidative phosphorylation (OXPHOS) system under environmental stress, has not been sufficiently addressed. In this study, we show that the putative YihA superfamily GTPase, Mrx8, is a bona fide mitochondrial protein required for Cox1 translation initiation and elongation during suboptimal growth condition at 16°C. Mrx8 was found in a complex with mitochondrial ribosomes, consistent with a role in protein synthesis. Cells expressing mutant Mrx8 predicted to be defective in guanine nucleotide binding and hydrolysis were compromised for robust cellular respiration. We show that the requirement of Pet309 and Mss51 for cellular respiration is not bypassed by overexpression of Mrx8 and vice versa. Consistently the ribosomal association of Mss51 is independent of Mrx8. Significantly, we find that GTPBP8, the human orthologue, complements the loss of cellular respiration in Δmrx8 cells and GTPBP8 localizes to the mitochondria in mammalian cells. This strongly suggests a universal role of the MRX8 family of proteins in regulating mitochondrial function.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , GTP Fosfohidrolasas/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Ribosomas Mitocondriales/metabolismo , Fosforilación Oxidativa , Biosíntesis de Proteínas , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Temperatura , Factores de Transcripción/metabolismoRESUMEN
The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.
Asunto(s)
Virus de la Hepatitis B/fisiología , Hepatitis B/metabolismo , Hepatitis B/virología , Transactivadores/metabolismo , Zinc/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Proteínas Recombinantes de Fusión , Transactivadores/química , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Monoclonal antibodies (mAbs) are an important class of biotherapeutics. Successful development of a mAb depends not only on its biological activity but also on its physicochemical properties, such as homogeneity and stability. mAb stability is affected by its formulation. Among the many techniques used to study the stability of mAbs, differential scanning fluorimetry (DSF) offers both excellent throughput and minimal material consumption. DSF measures the temperature of the protein unfolding transition (Tm) based on the change in fluorescence intensity of the environmentally sensitive dye SYPRO Orange. With DSF adapted to a 96-well plate format, we have shown that low-pH or high-salt concentrations decrease the thermal stability of mAb1, whereas some excipients, such as sucrose, polysorbate 80, and sodium phosphate, increase its stability. The basal fluorescence of SYPRO Orange was enhanced by the presence of detergents, limiting the use of this approach to diluted detergent solutions. Throughput of DSF can be increased further with the use of a 384-well plate. DSF thermograms are in good agreement with the melting profiles obtained by differential scanning calorimetry (DSC). The Tms determined by DSF and DSC were well correlated, with the former being on average lower by 3 °C.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Anticuerpos Monoclonales/químicaRESUMEN
1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea 14a (AR9281), a potent and selective soluble epoxide hydrolase inhibitor, was recently tested in a phase 2a clinical setting for its effectiveness in reducing blood pressure and improving insulin resistance in pre-diabetic patients. In a mouse model of diet induced obesity, AR9281 attenuated the enhanced glucose excursion following an intraperitoneal glucose tolerance test. AR9281 also attenuated the increase in blood pressure in angiotensin-II-induced hypertension in rats. These effects were dose-dependent and well correlated with inhibition of the sEH activity in whole blood, consistent with a role of sEH in the observed pharmacology in rodents.
Asunto(s)
Adamantano/análogos & derivados , Antihipertensivos/química , Inhibidores Enzimáticos/química , Epóxido Hidrolasas/antagonistas & inhibidores , Hipertensión/tratamiento farmacológico , Resistencia a la Insulina , Urea/análogos & derivados , Adamantano/química , Adamantano/farmacocinética , Adamantano/uso terapéutico , Administración Oral , Angiotensina II/farmacología , Animales , Antihipertensivos/farmacocinética , Antihipertensivos/uso terapéutico , Glucemia/análisis , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Epóxido Hidrolasas/metabolismo , Hipertensión/inducido químicamente , Ratones , Obesidad/tratamiento farmacológico , Ratas , Urea/química , Urea/farmacocinética , Urea/uso terapéuticoRESUMEN
OBJECTIVE: Epoxyeicosatrienoic acids (EETs) have been shown to have antiinflammatory effects and therefore may play a role in preventing vascular inflammatory and atherosclerotic diseases. Soluble epoxide hydrolase (s-EH) converts EETs into less bioactive dihydroxyeicosatrienoic acids. Thus, inhibition of s-EH can prevent degradation of EETs and prolong their effects. The present study aimed to test the hypothesis that inhibition of s-EH has vascular protective effects. METHODS AND RESULTS: Six-month-old apolipoprotein E-deficient mice were chronically infused with angiotensin II (1.44 mg/kg/d) for 4 weeks to induce abdominal aortic aneurysm (AAA), accelerate atherosclerosis development and carotid artery ligation-induced vascular remodeling. The mice were treated with a novel s-EH inhibitor, AR9276 (1.5 g/L in drinking water) or vehicle for 4 weeks. The results demonstrated that AR9276 significantly reduced the rate of AAA formation and atherosclerotic lesion area, but had no effect on ligation-induced carotid artery remodeling. These effects were associated with a reduction of serum lipid, IL-6, murine IL-8-KC, and IL-1alpha, and downregulation of gene expressions of ICAM-1, VCAM-1, and IL-6 in the arterial wall. CONCLUSIONS: The present data demonstrate that treatment with an s-EH inhibitor attenuates AAA formation and atherosclerosis development. The attendant downregulation of inflammatory mediators and lipid lowering effects may both contribute to the observed vascular protective effects.
Asunto(s)
Aneurisma de la Aorta Abdominal/prevención & control , Aterosclerosis/prevención & control , Dislipidemias/prevención & control , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Administración Oral , Angiotensina II , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/inducido químicamente , Aterosclerosis/enzimología , Aterosclerosis/patología , Disponibilidad Biológica , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/patología , Arterias Carótidas/cirugía , Colesterol/sangre , Modelos Animales de Enfermedad , Regulación hacia Abajo , Dislipidemias/inducido químicamente , Dislipidemias/enzimología , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacocinética , Epóxido Hidrolasas/metabolismo , Mediadores de Inflamación/sangre , Molécula 1 de Adhesión Intercelular/genética , Interleucina-1alfa/sangre , Interleucina-6/sangre , Interleucina-6/genética , Interleucina-8/sangre , Ligadura , Masculino , Ratones , Ratones Noqueados , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
In a search for more effective anti-diabetic treatment, we used a process coupling low-affinity biochemical screening with high-throughput co-crystallography in the design of a series of compounds that selectively modulate the activities of all three peroxisome proliferator-activated receptors (PPARs), PPARalpha, PPARgamma, and PPARdelta. Transcriptional transactivation assays were used to select compounds from this chemical series with a bias toward partial agonism toward PPARgamma, to circumvent the clinically observed side effects of full PPARgamma agonists. Co-crystallographic characterization of the lead molecule, indeglitazar, in complex with each of the 3 PPARs revealed the structural basis for its PPAR pan-activity and its partial agonistic response toward PPARgamma. Compared with full PPARgamma-agonists, indeglitazar is less potent in promoting adipocyte differentiation and only partially effective in stimulating adiponectin gene expression. Evaluation of the compound in vivo confirmed the reduced adiponectin response in animal models of obesity and diabetes while revealing strong beneficial effects on glucose, triglycerides, cholesterol, body weight, and other metabolic parameters. Indeglitazar has now progressed to Phase II clinical evaluations for Type 2 diabetes mellitus (T2DM).
Asunto(s)
Descubrimiento de Drogas/métodos , Hipoglucemiantes/uso terapéutico , PPAR gamma/agonistas , Receptores Activados del Proliferador del Peroxisoma/agonistas , Adipocitos/citología , Adiponectina/genética , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Diabetes Mellitus Experimental/tratamiento farmacológico , Humanos , Hipoglucemiantes/farmacología , Ratones , Obesidad/tratamiento farmacológico , PPAR gamma/genética , Receptores Activados del Proliferador del Peroxisoma/genética , Ratas , Activación Transcripcional/efectos de los fármacosRESUMEN
Steroidogenic factor-1 (SF-1) and liver receptor homologue-1 (LRH-1) belong to the fushi tarazu factor 1 subfamily of nuclear receptors. SF-1 is an essential factor for sex determination during development and regulates adrenal and gonadal steroidogenesis in the adult, whereas LRH-1 is a critical factor for development of endodermal tissues and regulates cholesterol and bile acid homeostasis. Regulatory ligands are unknown for SF-1 and LRH-1. A reported mouse LRH-1 structure revealed an empty pocket in a region commonly occupied by ligands in the structures of other nuclear receptors, and pocket-filling mutations did not alter the constitutive activity observed. Here we report the crystal structures of the putative ligand-binding domains of human SF-1 at 2.1-A resolution and human LRH-1 at 2.5-A resolution. Both structures bind a coactivator-derived peptide at the canonical activation-function surface, thus adopting the transcriptionally activating conformation. In human LRH-1, coactivator peptide binding also occurs to a second site. We discovered in both structures a phospholipid molecule bound in a pocket of the putative ligand-binding domain. MS analysis of the protein samples used for crystallization indicated that the two proteins associate with a range of phospholipids. Mutations of the pocket-lining residues reduced the transcriptional activities of SF-1 and LRH-1 in mammalian cell transfection assays without affecting their expression levels. These results suggest that human SF-1 and LRH-1 may be ligand-binding receptors, although it remains to be seen if phospholipids or possibly other molecules regulate SF-1 or LRH-1 under physiological conditions.
Asunto(s)
Proteínas de Unión al ADN/ultraestructura , Modelos Moleculares , Receptores Citoplasmáticos y Nucleares/ultraestructura , Factores de Transcripción/ultraestructura , Secuencia de Aminoácidos , Línea Celular , Cristalografía , Citomegalovirus , Proteínas de Unión al ADN/genética , Vectores Genéticos , Proteínas de Homeodominio , Humanos , Ligandos , Espectrometría de Masas , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/genética , Factor Esteroidogénico 1 , Factores de Transcripción/genética , TransfecciónRESUMEN
Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC(50)s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other beta-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC(50)s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.