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The plasma protein α1-acid glycoprotein (AGP) primarily affects the pharmacokinetics of basic drugs. There are two AGP variants in humans, A and F1*S, exhibiting distinct drug-binding selectivity. Elucidation of the drug-binding selectivity of human AGP variants is essential for drug development and personalized drug therapy. Herein, we aimed to establish the contribution of amino acids 112 and 114 of human AGP to drug-binding selectively. Both amino acids are located in the drug-binding region and differ between the variants. Phe112/Ser114 of the A variant and its equivalent residues in the F1*S variant (Leu112/Phe114) were swapped with each other. Binding experiments were then conducted using the antiarrhythmic drug disopyramide, which selectively binds to the A variant. A significant decrease in the bound fraction was observed in each singly mutated A protein (Phe112Leu or Ser114Phe). Moreover, the bound fraction of the double A mutant (Phe112Leu/Ser114Phe) was decreased to that of wild-type F1*S. Intriguingly, the double F1*S mutant (Leu112Phe/Phe114Ser), in which residues were swapped with those of the A variant, showed only partial restoration in binding. The triple F1*S mutant (Leu112Phe/Phe114Ser/Asp115Tyr), where position 115 is thought to contribute to the difference in pocket size between variants, showed a further recovery in binding to 70% of that of wild-type A. These results were supported by thermodynamic analysis and acridine orange binding, which selectively binds the A variant. Together, these data indicate that, in addition to direct interaction with Phe112 and Ser114, the binding pocket size contributed by Tyr115 is important for the drug-binding selectivity of the A variant.
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Orosomucoide , Unión Proteica , Orosomucoide/metabolismo , Orosomucoide/genética , Orosomucoide/química , Humanos , Sitios de Unión , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Tirosina/química , Tirosina/metabolismo , Tirosina/genética , Mutación , Serina/metabolismo , Serina/genética , Serina/química , Antiarrítmicos/química , Antiarrítmicos/metabolismoRESUMEN
Objectives: The efficacy of endovascular aneurysm repair (EVAR) against abdominal aortic aneurysm (AAA) in younger patients remains unknown. Hence, the current study aimed to investigate whether the aneurysm-related mortality rate of EVAR is acceptable among patients aged ≤70 years. Methods: Among 644 patients, 148 underwent EVAR (EVAR group), and 496 received open surgical repair (OSR group). The cumulative incidence rates of aneurysm-related death, any intervention, and serious aneurysm-related events after AAA repair were evaluated using the cumulative incidence function in the presence of competing risks. Results: The EVAR group had higher prevalences of several comorbidities, and overall survival for the EVAR group was significantly inferior to that of the OSR group. The cumulative incidence rates of aneurysm-related death, any intervention, and serious aneurysm-related events at 5 years were 1.5%, 11.7%, and 6.4% in the EVAR group and 1.3%, 5.3%, and 5.9% in the OSR group, respectively. EVAR was not a significant prognostic factor of aneurysm-related mortality and serious aneurysm-related events. However, it was an independent poor prognostic factor of any intervention. Conclusion: EVAR was not a significant prognostic factor of aneurysm-related mortality and serious aneurysm-related events. Therefore, it demonstrated acceptable procedure-related long-term outcomes, at least in high-risk young patients.
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Nuclear factor κB (NF-κB) is activated by various inflammatory and infectious molecules and is involved in immune responses. It has been elucidated that ADP-ß-D-manno-heptose (ADP-Hep), a metabolite in gram-negative bacteria, activates NF-κB through alpha-kinase 1 (ALPK1)-TIFA-TRAF6 signaling. ADP-Hep stimulates the kinase activity of ALPK1 for TIFA phosphorylation. Complex formation between phosphorylation-dependent TIFA oligomer and TRAF6 promotes the polyubiquitination of TRAF6 for NF-κB activation. TIFAB, a TIFA homolog lacking a phosphorylation site and a TRAF6 binding motif, is a negative regulator of TIFA-TRAF6 signaling and is implicated in myeloid diseases. TIFAB is indicated to regulate TIFA-TRAF6 signaling through interactions with TIFA and TRAF6; however, little is known about its biological function. We demonstrated that TIFAB forms a complex not with the TIFA dimer, an intrinsic form of TIFA involved in NF-κB activation, but with monomeric TIFA. The structural analysis of the TIFA/TIFAB complex and the biochemical and cell-based analyses showed that TIFAB forms a stable heterodimer with TIFA, inhibits TIFA dimer formation, and suppresses TIFA-TRAF6 signaling. The resultant TIFA/TIFAB complex is a "pseudo-TIFA dimer" lacking the phosphorylation site and TRAF6 binding motif in TIFAB and cannot form the orderly structure as proposed for the phosphorylated TIFA oligomer involved in NF-κB activation. This study elucidated the molecular and structural basis for the regulation of TIFA-TRAF6 signaling by TIFAB.
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FN-kappa B , Factor 6 Asociado a Receptor de TNF , Factor 6 Asociado a Receptor de TNF/genética , Transducción de Señal , Inmunidad Innata , Fosforilación , PolímerosRESUMEN
AIMS: Paraoxonase 1 (PON1) binds to high-density lipoprotein (HDL) and protects against atherosclerosis. However, the relationship between functional PON1 Q192R polymorphism, which is associated with the hydrolysis of paraoxon (POXase activity) and atherosclerotic cardiovascular disease (ASCVD), remains controversial. As the effect of PON1 Q192R polymorphism on the HDL function is unclear, we investigated the relationship between this polymorphism and the cholesterol efflux capacity (CEC), one of the biological functions of HDL, in association with the PON1 activity. METHODS: The relationship between PON1 Q192R polymorphisms and CEC was investigated retrospectively in 150 subjects without ASCVD (50 with the PON1 Q/Q genotype, 50 with the Q/R genotype, and 50 with the R/R genotype) who participated in a health screening program. The POXase and arylesterase (AREase: hydrolysis of aromatic esters) activities were used as measures of the PON1 activity. RESULTS: The AREase activity was positively correlated with CEC independent of the HDL cholesterol levels. When stratified by the PON1 Q192R genotype, the POXase activity was also positively correlated with CEC independent of HDL cholesterol. PON1 Q192R R/R genotype carriers had a lower CEC than Q/Q or Q/R genotype carriers, despite having a higher POXase activity. Moreover, in a multiple regression analysis, the PON1 Q192R genotype was associated with the degree of CEC, independent of the HDL cholesterol and POXase activity. CONCLUSIONS: The PON1 Q192R R allele is associated with reduced CEC in Japanese people without ASCVD. Further studies on the impact of this association on the severity of atherosclerosis and ASCVD development are thus called for.
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Introduction: In the context of double-valve surgery for elderly high-risk patients involving both the aortic and mitral valves, a clinically significant problem has been that no clear criteria or surgical strategies have been reported for the selection of mitral valve plasty (MVP) or mitral valve replacement (MVR) for mitral valve disease management during surgical aortic valve replacement (SAVR) to achieve better clinical outcomes. This study investigated valve durability and survival using our surgical strategy for mitral valve disease with concomitant SAVR in elderly patients. Methods: Eighty-six patients aged > 65 years (mean 75 years) who underwent a double-valve procedure for mitral valve surgery with concomitant SAVR from 2010 to 2022 were reviewed. Our surgical strategy for mitral valve disease with concomitant SAVR for the elderly patients was as follows: MVP was selected for patients in whom mitral valve disease was expected to be controlled with simple surgical procedures (n = 47), otherwise MVR was selected (n = 39). Results: The hospital mortality rate was 8% (n = 7). The mean follow-up was 4.9 (0-12.3) years. And the 10-year survival rate was 62%. The 10-year freedom from aortic valve reoperation rate was 95%. No mitral valve reintervention was performed during follow-up. Echocardiographic follow-up demonstrated freedom from at least moderate mitral regurgitation in 86% of cases at 10 years. Conclusion: In double-valve surgery for elderly high-risk patients, appropriate selection of the mitral valve procedure with concomitant SAVR provided better early and long-term survival and valve durability. This surgical strategy may be beneficial in elderly patients with combined aortic and mitral valve disease.
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Hepatitis is an inflammation of the liver caused by the inadequate elimination of reactive oxygen species (ROS) derived from Kupffer cells. Edaravone is clinically used as an antioxidant but shows poor liver distribution. Herein, we report on the design of a Kupffer cell-oriented nanoantioxidant based on a disulfide cross-linked albumin nanoparticle containing encapsulated edaravone (EeNA) as a therapeutic for the treatment of hepatitis. Since the edaravone is bound to albumin, this results in a soluble and stable form of edaravone in water. Exchanging the intramolecular disulfide bonds to intermolecular disulfide bridges of albumin molecules allowed the preparation of a redox responsive albumin nanoparticle that is stable in the blood circulation but can release drugs into cells. Consequently, EeNA was fabricated by the nanoscale self-assembly of edaravone and albumin nanoparticles without the additives that are contained in commercially available edaravone preparations. EeNA retained its nanostructure under serum conditions, but the encapsulated edaravone was released efficiently under intracellular reducing conditions in macrophages. The EeNA was largely distributed in the liver and subsequently internalized into Kupffer cells within 60 min after injection in a concanavalin-A-induced hepatitis mouse. The survival rate of the hepatitis mice was significantly improved by EeNA due to the suppression of liver necrosis and oxidative stress by scavenging excessive ROS. Moreover, even through the postadministration, EeNA showed an excellent hepatoprotective action as well. In conclusion, EeNA has the potential for use as a nanotherapeutic against various types of hepatitis because of its Kupffer cell targeting ability and redox characteristics.
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Hepatitis , Nanopartículas , Animales , Ratones , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Edaravona , Hepatitis/tratamiento farmacológico , Albúminas/metabolismo , Oxidación-Reducción , Nanopartículas/química , DisulfurosRESUMEN
In recent years, protein drug development has gained momentum, and simple and facile controlled-release systems without loss of activity are required. Herein, we developed a sustained-release system for protein drugs by exploiting the "astringency" mechanism, namely insoluble precipitate formation by interacting with tannic acid. Tannic acid formed insoluble precipitates with various protein drugs, such as nisin, insulin, lysozyme, ovalbumin, hyaluronidase, and human immunoglobulin G, through hydrophobic interactions and hydrogen bonds. The lysozyme/tannic acid complex retained in vitro lytic activity. Precipitates of the insulin/tannic acid complex prolonged hypoglycemic effects without loss of activity after subcutaneous administration. The ovalbumin/tannic acid complex enhanced anti-ovalbumin antibody production induced by ovalbumin, which may be attributed to its sustained-release profile. Accordingly, tannic acid is useful as a simple and user-friendly drug delivery system for protein drugs.
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Insulinas , Muramidasa , Humanos , Preparaciones de Acción Retardada , Taninos/química , OvalbúminaRESUMEN
Human MutT homolog 1 (MTH1), also known as Nudix-type motif 1 (NUDT1), hydrolyzes 8-oxo-dGTP and 2-oxo-dATP with broad substrate recognition and has attracted attention in anticancer therapeutics. Previous studies on MTH1 have proposed that the exchange of the protonation state between Asp119 and Asp120 is essential for the broad substrate recognition of MTH1. To understand the relationship between protonation states and substrate binding, we determined the crystal structures of MTH1 at pH 7.7-9.7. With increasing pH, MTH1 gradually loses its substrate-binding ability, indicating that Asp119 is deprotonated at pH 8.0-9.1 in 8-oxo-dGTP recognition and Asp120 is deprotonated at pH 8.6-9.7 in 2-oxo-dATP recognition. These results confirm that MTH1 recognizes 8-oxo-dGTP and 2-oxo-dATP by exchanging the protonation state between Asp119 and Asp120 with higher pKa .
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Monoéster Fosfórico Hidrolasas , Pirofosfatasas , Humanos , Pirofosfatasas/química , Pirofosfatasas/metabolismo , Monoéster Fosfórico Hidrolasas/química , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Hidrolasas NudixRESUMEN
Escherichia coli MutT prevents mutations by hydrolyzing mutagenic 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) in the presence of Mg2+ or Mn2+ ions. MutT is one of the most studied enzymes in the nucleoside diphosphate-linked moiety X (Nudix) hydrolase superfamily, which is widely distributed in living organisms. However, the catalytic mechanisms of most Nudix hydrolases, including two- or three-metal-ion mechanisms, are still unclear because these mechanisms are proposed using the structures mimicking the reaction states, such as substrate analog complexes. Here, we visualized the hydrolytic reaction process of MutT by time-resolved X-ray crystallography using a biological substrate, 8-oxo-dGTP, and an active metal ion, Mn2+. The reaction was initiated by soaking MutT crystals in a MnCl2 solution and stopped by freezing the crystals at various time points. In total, five types of intermediate structures were refined by investigating the time course of the electron densities in the active site as well as the anomalous signal intensities of Mn2+ ions. The structures and electron densities show that three Mn2+ ions bind to the Nudix motif of MutT and align the substrate 8-oxo-dGTP for catalysis. Accompanied by the coordination of the three Mn2+ ions, a water molecule, bound to a catalytic base, forms a binuclear Mn2+ center for nucleophilic substitution at the ß-phosphorus of 8-oxo-dGTP. The reaction condition using Mg2+ also captured a structure in complex with three Mg2+ ions. This study provides the structural details essential for understanding the three-metal-ion mechanism of Nudix hydrolases and proposes that some of the Nudix hydrolases share this mechanism.
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Proteínas de Escherichia coli , Escherichia coli , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Mutágenos , Monoéster Fosfórico Hidrolasas/metabolismo , Pirofosfatasas/metabolismo , Hidrolasas NudixRESUMEN
Three new diterpenes, stellejasmins A (1) and B (2) and 12-O-benzoylphorbol-13-heptanoate (3), were isolated from the roots of Stellera chamaejasme L. The structures of 1-3 were elucidated by extensive NMR and mass spectroscopic analyses. Compounds 1 and 2 are the first derivatives containing a hydroxy group at C-2 in the family of daphnane and tigliane diterpenes. The presence of a chlorine atom in 1 is unique in the plant metabolite. Compound 3 has an odd-number acyl group, which is biosynthetically notable. Human immunodeficiency virus (HIV) LTR-driven transcription activity was tested with 1-3 and 17 known diterpenes isolated from S. chamaejasme L. and Wikstroemia retusa A.Gray. Among these, gnidimacrin (4), stelleralide A (5), and wikstroelide A (20) were highly potent, with EC50 values of 0.14, 0.33, and 0.39 nM, respectively. The structure-activity relationship (SAR) was investigated using 20 natural and eight synthetic diterpenes. This is the first SAR study on natural daphnane and tigliane diterpenes.
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Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Diterpenos/síntesis química , Diterpenos/farmacología , VIH/efectos de los fármacos , Forboles/química , Latencia del Virus/efectos de los fármacos , Diterpenos/química , Modelos Moleculares , Simulación del Acoplamiento Molecular , Forboles/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Raíces de Plantas/química , Relación Estructura-Actividad , Thymelaeaceae/química , Wikstroemia/químicaRESUMEN
Mammalian MutY homologue (MUTYH) is an adenine DNA glycosylase that excises adenine inserted opposite 8-oxoguanine (8-oxoG). The inherited variations in human MUTYH gene are known to cause MUTYH-associated polyposis (MAP), which is associated with colorectal cancer. MUTYH is involved in base excision repair (BER) with proliferating cell nuclear antigen (PCNA) in DNA replication, which is unique and critical for effective mutation-avoidance. It is also reported that MUTYH has a Zn-binding motif in a unique interdomain connector (IDC) region, which interacts with Rad9-Rad1-Hus1 complex (9-1-1) in DNA damage response, and with apurinic/apyrimidinic endonuclease 1 (APE1) in BER. However, the structural basis for the BER pathway by MUTYH and its interacting proteins is unclear. Here, we determined the crystal structures of complexes between mouse MUTYH and DNA, and between the C-terminal domain of mouse MUTYH and human PCNA. The structures elucidated the repair mechanism for the A:8-oxoG mispair including DNA replication-coupled repair process involving MUTYH and PCNA. The Zn-binding motif was revealed to comprise one histidine and three cysteine residues. The IDC, including the Zn-binding motif, is exposed on the MUTYH surface, suggesting its interaction modes with 9-1-1 and APE1, respectively. The structure of MUTYH explains how MAP mutations perturb MUTYH function.
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ADN Glicosilasas/química , Adenina , Poliposis Adenomatosa del Colon/genética , Secuencias de Aminoácidos , Animales , ADN/química , ADN Glicosilasas/genética , Reparación del ADN , Replicación del ADN , Guanina/análogos & derivados , Humanos , Ratones , Modelos Moleculares , Mutación , Antígeno Nuclear de Célula en Proliferación/química , ZincRESUMEN
Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by non-enzymatic reaction between reducing-sugar and Arg/Lys in proteins and are involved in various diabetic complications. GA-pyridine is derived from glycolaldehyde and is one of the most cytotoxic AGEs. Here, we established a single-chain Fv (scFv) antibody against GA-pyridine, 73MuL9-scFv, and examined the details of its specificity and antigen recognition by using various techniques involving biophysics, chemical biology and structural biology. We also synthesized several compounds that differ slightly in regard to the position and number of GA-pyridine substituent groups, and revealed that GA-pyridine was specifically bound to 73MuL9-scFv. Thermodynamic analysis revealed that the association of GA-pyridine to 73MuL9-scFv was an exothermic and enthalpy driven reaction, and thus that the antigen recognition involved multiple specific interactions. Crystallographic analysis of the Fv fragment of 73MuL9-scFv revealed that several CH-π and hydrogen bond interactions took place between the Fv-fragment and GA-pyridine, which was consistent with the results of thermodynamic analysis. Further studies using 73MuL9-scFv as a tool to clarify the relevance of GA-pyridine to diabetic complications are warranted.
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Productos Finales de Glicación Avanzada/inmunología , Piridinas/inmunología , Anticuerpos de Cadena Única/metabolismo , Acetaldehído/análogos & derivados , Acetaldehído/química , Acetaldehído/inmunología , Secuencia de Aminoácidos , Antígenos/química , Antígenos/metabolismo , Biofisica , Cristalografía/métodos , Productos Finales de Glicación Avanzada/química , Humanos , Enlace de Hidrógeno , Piridinas/química , Anticuerpos de Cadena Única/química , TermodinámicaRESUMEN
The E3 ubiquitin ligase RAD18 mono-ubiquitinates PCNA to promote bypass of replication fork-stalling DNA lesions. On the other hand, RAD18 also contributes to DNA double-strand break (DSB) repair. RAD18 is recruited to ionizing radiation (IR)-induced DSB and colocalizes with ubiquitinated chromatin proteins. RAD18 interacts with the ubiquitinated chromatin proteins via its ubiquitin-binding Zinc finger (UBZ) domain and is proposed to propagate DNA DSB signalling and recruit DNA repair proteins. We found that purified human RAD18 protein complexed with RAD6B (RAD6B-RAD18) catalyzes mono- and poly-ubiquitination of histone H2A in vitro while UBZ domain-mutated RAD18 complexed with RAD6B protein catalyzes mono- but not poly-ubiquitination of histone H2A. Human RAD18-/-cells synchronized at the G1 phase show a reduced signal of ubiquitinated protein in chromatin after IR when compared to that of wild-type control cells. The reduced signal of ubiquitinated protein in RAD18-/-cells is rescued by the introduction of RAD18 cDNA but to a lesser extent by the introduction of cDNA coding RAD18 lacking UBZ domain. Taken together, these results indicate that RAD18 mediates DSB-induced ubiquitination of chromatin protein during the G1 phase.
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Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células Cultivadas , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/deficiencia , Humanos , Ubiquitina-Proteína Ligasas/deficiencia , UbiquitinaciónRESUMEN
The blood-brain barrier (BBB) prevents the permeability of drugs into the brain, and as such limits the management of various brain diseases. To overcome this barrier, drug-encapsulating nanoparticles or vesicles, drug conjugates, and other types of drug delivery systems (DDSs) have been developed. However, the brain-targeting ability of nanoparticles or vesicles is still insufficient. Recently, among the various brain-targeting ligands previously studied for facilitating transcellular BBB transport, several sugar-appended nanocarriers for brain delivery were identified. Meanwhile, cyclodextrins (CyDs) have been used as nanocarriers for drug delivery since they can encapsulate hydrophobic compounds with high biocompatibility. Therefore, in this study, we created various sugar-appended ß-cyclodextrins (ß-CyDs) to discover novel brain-targeting ligands. As a result, of the six sugar-appended CyDs, lactose-appended ß-CyD (Lac-ß-CyD) showed greater cellular uptake in hCMEC/D3 cells, human brain microvascular endothelial cells, than other sugar-appended ß-CyDs did. In addition, the permeability of Lac-ß-CyD within the in vitro human BBB model was greater than that of other sugar-appended ß-CyDs. Moreover, Lac-ß-CyD significantly accumulated in the mouse brain after intravenous administration. Thus, Lac-ß-CyD efficiently facilitated the accumulation of the model drug into the mouse brain. These findings suggest that Lac-ß-CyD has the potential to be a novel carrier for drugs across the BBB.
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Ciclodextrinas , beta-Ciclodextrinas , Encéfalo , Células Endoteliales , LactosaRESUMEN
HIV-1 often acquires drug-resistant mutations in spite of the benefits of antiretroviral therapy (ART). HIV-1 integrase (IN) is essential for the concerted integration of HIV-1 DNA into the host genome. IN further contributes to HIV-1 RNA binding, which is required for HIV-1 maturation. Non-catalytic-site integrase inhibitors (NCINIs) have been developed as allosteric IN inhibitors, which perform anti-HIV-1 activity by a multimodal mode of action such as inhibition of the IN-lens epithelium-derived growth factor (LEDGF)/p75 interaction in the early stage and disruption of functional IN multimerization in the late stage of HIV-1 replication. Here, we show that IN undergoes an adaptable conformational change to escape from NCINIs. We observed that NCINI-resistant HIV-1 variants have accumulated 4 amino acid mutations by passage 26 (P26) in the IN-encoding region. We employed high-performance liquid chromatography (HPLC), thermal stability assays, and X-ray crystallographic analysis to show that some amino acid mutations affect the stability and/or dimerization interface of the IN catalytic core domains (CCDs), potentially resulting in the severely decreased multimerization of full-length IN proteins (IN undermultimerization). This undermultimerized IN via NCINI-related mutations was stabilized by HIV-1 RNA and restored to the same level as that of wild-type HIV-1 in viral particles. Recombinant HIV-1 clones with IN undermultimerization propagated similarly to wild-type HIV-1. Our study revealed that HIV-1 can eventually counteract NCINI-induced IN overmultimerization by IN undermultimerization as one of the escape mechanisms. Our findings provide information on the understanding of IN multimerization with or without HIV-1 RNA and may influence the development of anti-HIV-1 strategies.IMPORTANCE Understanding the mechanism of HIV-1 resistance to anti-HIV-1 drugs could lead to the development of novel drugs with increased efficiency, resulting in more effective ART. ART composed of more potent and long-acting anti-HIV-1 drugs can greatly improve drug adherence and also provide HIV-1 prevention such as preexposure prophylaxis. NCINIs with a multimodal mode of action exert potent anti-HIV-1 effects through IN overmultimerization during HIV-1 maturation. However, HIV-1 can acquire some mutations that cause IN undermultimerization to alleviate NCINI-induced IN overmultimerization. This undermultimerized IN was efficiently stabilized by HIV-1 RNA and restored to the same level as that of wild-type HIV-1. Our findings revealed that HIV-1 eventually acquires such a conformational escape reaction to overcome the unique NCINI actions. The investigation into drug-resistant mutations associated with HIV-1 protein multimerization may facilitate the elucidation of its molecular mechanism and functional multimerization, allowing us to develop more potent anti-HIV-1 drugs and unique treatment strategies.
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Regulación Alostérica/efectos de los fármacos , Fármacos Anti-VIH/farmacología , Reacción de Fuga/efectos de los fármacos , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Regulación Alostérica/genética , Células HEK293 , Infecciones por VIH/tratamiento farmacológico , Integrasa de VIH/metabolismo , Inhibidores de Integrasa VIH/química , VIH-1/genética , VIH-1/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Mutación , Multimerización de Proteína/efectos de los fármacos , Proteínas Recombinantes , Factores de Transcripción , Virión/química , Virión/genética , Replicación Viral/efectos de los fármacosRESUMEN
TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA), originally identified as an adaptor protein of TRAF6, has recently been shown to be involved in innate immunity, induced by a pathogen-associated molecular pattern (PAMP). ADP-ß-D-manno-heptose, a newly identified PAMP, binds to alpha-kinase 1 (ALPK1) and activates its kinase activity to phosphorylate TIFA. Phosphorylation triggers TIFA oligomerisation and formation of a subsequent TIFA-TRAF6 oligomeric complex for ubiquitination of TRAF6, eventually leading to NF-κB activation. However, the structural basis of TIFA-dependent TRAF6 signalling, especially oligomer formation of the TIFA-TRAF6 complex remains unknown. In the present study, we determined the crystal structures of mouse TIFA and two TIFA mutants-Thr9 mutated to either Asp or Glu to mimic the phosphorylation state-to obtain the structural information for oligomer formation of the TIFA-TRAF6 complex. Crystal structures show the dimer formation of mouse TIFA to be similar to that of human TIFA, which was previously reported. This dimeric structure is consistent with the solution structure obtained from small angle X-ray scattering analysis. In addition to the structural analysis, we examined the molecular assembly of TIFA and the TIFA-TRAF6 complex by size-exclusion chromatography, and suggested a model for the TIFA-TRAF6 signalling complex.
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Inmunidad Innata/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/ultraestructura , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Ratones , FN-kappa B/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , UbiquitinaciónRESUMEN
Surgical strategy for significant carotid artery stenosis complicated with severe aortic valve stenosis is still controversial. Herein, we report a case of 80-year-old female in whom 78% stenosis by the NASCET criteria in left internal carotid artery was pointed out during preoperative checkup for symptomatic severe aortic stenosis. Carotid endarterectomy was done concomitantly with aortic valve replacement. No neurological complication occurred perioperatively.
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Estenosis de la Válvula Aórtica/cirugía , Válvula Aórtica/cirugía , Estenosis Carotídea/cirugía , Endarterectomía Carotidea/métodos , Accidente Cerebrovascular/etiología , Anciano de 80 o más Años , Estenosis Carotídea/complicaciones , Femenino , Prótesis Valvulares Cardíacas , Humanos , Reemplazo de la Válvula Aórtica TranscatéterRESUMEN
BACKGROUND: We evaluated clinical outcomes of left ventricular assist device (LVAD) support in patients with or without severe right heart failure, in order to determine what kind of organ allocation system could help severe biventricular failure patients to be safely bridged to heart transplantation (HTx), even in Japan where the waiting time for HTx is extremely long. MethodsâandâResults: One hundred and seventy consecutive patients who were implanted with continuous-flow LVAD at the present institution were included in this study. The patients were divided into 2 groups: 158 patients with isolated LVAD (group-LVF) and 12 patients who required long-term mechanical or inotropic right heart support (group-BVF). Post-LVAD survival in group-BVF was significantly worse than in group-LVF (P<0.0001). Given that many patients in group-BVF died between 1 and 2 years after LVAD implantation, Kaplan-Meier survival curve simulation was carried out under the condition that all the patients in group-BVF who died on LVAD support >1 year after LVAD implantation had received HTx at 365 days after LVAD implantation and survived thereafter. In this simulation, no significant difference in survival was seen between the groups (P=0.2424). CONCLUSIONS: A new allocation system that allows severe right heart failure patients to receive HTx at around 1 year would enable rescue of the patients with severe right heart failure.
Asunto(s)
Insuficiencia Cardíaca/terapia , Corazón Auxiliar , Disfunción Ventricular Izquierda/terapia , Adulto , Terapia de Resincronización Cardíaca/métodos , Femenino , Insuficiencia Cardíaca/cirugía , Trasplante de Corazón , Humanos , Masculino , Persona de Mediana Edad , Contracción Miocárdica , Adulto JovenRESUMEN
SP7/Osterix (OSX) is a master regulatory transcription factor that activates a variety of genes during differentiation of osteoblasts. However, the influence of post-translational modifications on the regulation of its transactivation activity is largely unknown. Here, we report that sirtuins, which are NAD(+)-dependent deacylases, regulate lysine deacylation-mediated transactivation of OSX. Germline Sirt7 knockout mice develop severe osteopenia characterized by decreased bone formation and an increase of osteoclasts. Similarly, osteoblast-specific Sirt7 knockout mice showed attenuated bone formation. Interaction of SIRT7 with OSX leads to the activation of transactivation by OSX without altering its protein expression. Deacylation of lysine (K) 368 in the C-terminal region of OSX by SIRT7 promote its N-terminal transactivation activity. In addition, SIRT7-mediated deacylation of K368 also facilitates depropionylation of OSX by SIRT1, thereby increasing OSX transactivation activity. In conclusion, our findings suggest that SIRT7 has a critical role in bone formation by regulating acylation of OSX.
Asunto(s)
Enfermedades Óseas Metabólicas/genética , Lisina/metabolismo , Osteoblastos/metabolismo , Sirtuinas/genética , Factor de Transcripción Sp7/genética , Activación Transcripcional , Acilación , Animales , Densidad Ósea , Enfermedades Óseas Metabólicas/metabolismo , Enfermedades Óseas Metabólicas/patología , Diferenciación Celular , Línea Celular , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Osteogénesis/genética , Transducción de Señal , Sirtuinas/deficiencia , Factor de Transcripción Sp7/metabolismoRESUMEN
Anammox is a bacterial energy metabolic process that forms N2 gas from nitrite and ammonium ions. The enzymatic mechanisms of anammox have been gradually revealed; however, the electron transport chain in anammox bacteria remains poorly understood. In the present study, we purified and characterized two low-molecular-weight c-type cytochromes from an enriched culture of the anammox bacterium strain, KSU-1. Their genes, KSU1_B0428 and KSU1_C0855, were identified in the KSU-1 genome, and their recombinant proteins were characterized. KSU1_B0428 is a typical c-type cytochrome with a His/Met coordinated heme, acting as an electron transfer protein. In contrast, KSU1_C0855 could not be assigned as a known cytochrome and its heme was suggested to have an uncommon axial ligand set. Crystal structural analyses of C0855 clearly showed that its heme iron is coordinated by His15 as a fifth ligand. Moreover, the sixth coordination site is occupied by the aromatic ring of Tyr60, and an unassignable electron density that is inseparable with that of aromatic carbon of Tyr60 was found. The additional electron density was assigned to an O atom by molecular mass analyses. Therefore, Tyr60 would be chemically modified to 3,4-dihydroxyphenylalanine and bound to the Fe atom. We revealed that an anammox bacterium strain KSU-1 expresses a novel cytochrome c having an unprecedented His/3,4-dihydroxyphenylalanine coordinating heme. The expression of the novel c-type cytochrome might be required for the redox reaction of the anammox process.