RESUMEN
BACKGROUND: The prevalence of anxiety in patients undergoing total knee arthroplasty (TKA) and its association with postoperative functions are well known; however, the levels of anxiety or anxiety-related characteristics are unknown. This study aimed to investigate the prevalence of clinically significant state anxiety in geriatric patients undergoing TKA for osteoarthritis (OA) of the knee and to evaluate the anxiety-related characteristics experienced by these patients pre- and post-operatively. METHODS: This retrospective observational study recruited patients who had undergone TKA for knee OA using general anesthesia between February 2020 and August 2021. The study participants were geriatric patients older than 65 years who had moderate or severe OA. We evaluated patient characteristics including age, sex, body mass index, smoking status, hypertension, diabetes, and cancer. We assessed their levels of anxiety status using the STAI-X which comprises 20-item scales. Clinically meaningful state anxiety was defined as a total score of 52 or higher. An independent Student's t-test was used to determine differences of STAI score between subgroups in terms of patient characteristics. And patients were asked to complete questionnaires, which assessed four areas: (1) the main cause of anxiety; (2) the most helpful factor in overcoming anxiety before surgery; (3) the most helpful factor in reducing anxiety after surgery; and (4) the most anxious moment during the entire process. RESULTS: The mean STAI score of patients who underwent TKA was 43.0 points and 16.4% of patients experienced clinically significant state anxiety. The current smoking status affect STAI score and the proportion of patients with clinically meaningful state anxiety. The most common cause of preoperative anxiety was the surgery itself. Overall, 38% of patients reported that they experienced the greatest level of anxiety when the surgeon had recommended TKA in the outpatient clinic. The trust in the medical staff before surgery and the surgeon's explanations after surgery helped the most in reducing anxiety. CONCLUSIONS: One in six patients before TKA experience clinically meaningful state anxiety, and about 40% of patients experience anxiety from the time they are recommended for surgery. Patients tended to overcome anxiety before TKA through trust in the medical staff, and the surgeon's explanations after surgery was found to be helpful in reducing anxiety.
Asunto(s)
Artroplastia de Reemplazo de Rodilla , Osteoartritis de la Rodilla , Humanos , Anciano , Artroplastia de Reemplazo de Rodilla/efectos adversos , Ansiedad/diagnóstico , Ansiedad/epidemiología , Ansiedad/etiología , Trastornos de Ansiedad , Articulación de la Rodilla , Osteoartritis de la Rodilla/epidemiología , Osteoartritis de la Rodilla/cirugíaRESUMEN
Cupriavidus necator H16 is a gram-negative chemolithoautotrophic bacterium that has been extensively studied for biosynthesis and biodegradation of polyhydroxyalkanoate (PHA) plastics. To improve our understanding of fatty acid metabolism for PHA production, we determined the crystal structure of multi-functional enoyl-CoA hydratase from Cupriavidus necator H16 (CnFadB). The predicted model of CnFadB created by AlphaFold was used to solve the phase problem during determination of the crystal structure of the protein. The CnFadB structure consists of two distinctive domains, an N-terminal enol-CoA hydratase (ECH) domain and a C-terminal 3-hydroxyacyl-CoA dehydrogenase (HAD) domain, and the substrate- and cofactor-binding modes of these two functional domains were identified. Unlike other known FadB enzymes that exist as dimers complexed with FadA, CnFadB functions as a monomer without forming a complex with CnFadA. Small angle X-ray scattering (SAXS) measurement further proved that CnFadB exists as a monomer in solution. The non-sequential action of FadA and FadB in C. necator appears to affect ß-oxidation and PHA synthesis/degradation.
Asunto(s)
Cupriavidus necator , Polihidroxialcanoatos , Cupriavidus necator/metabolismo , Polihidroxialcanoatos/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Enoil-CoA Hidratasa/metabolismo , Ácidos Grasos/metabolismo , Plásticos/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasa/metabolismo , Coenzima A/metabolismoRESUMEN
Cerebrospinal fluid (CSF) amyloid-beta 1-42 (Aß1-42) and amyloid positron emission tomography (PET) are the 2 main Alzheimer disease amyloid biomarkers that have been validated in neuropathologically confirmed Alzheimer disease cases. Although many studies have shown concordance of amyloid positivity or negativity between CSF Aß1-42 and amyloid PET, several studies also reported discrepancies between these 2 Aß biomarkers. We conducted a comparison of CSF Aß1-42 level, amyloid PET, and autopsy findings in a case with progressive supranuclear palsy in which biomarker acquisition and postmortem pathologic examination were conducted almost at the same time. Our case with antemortem CSF Aß1-42 (+)/amyloid PET (-) who was pathologically confirmed with Aß pathology in the cerebral cortex may indicate CSF Aß1-42 is more sensitive for assessing in vivo Aß than amyloid PET.
Asunto(s)
Péptidos beta-Amiloides/líquido cefalorraquídeo , Autopsia , Biomarcadores/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , Parálisis Supranuclear Progresiva/líquido cefalorraquídeo , Parálisis Supranuclear Progresiva/patología , Anciano , Encéfalo/patología , Humanos , Masculino , Tomografía de Emisión de Positrones , Parálisis Supranuclear Progresiva/diagnóstico por imagenRESUMEN
In metabolic engineering and synthetic biology fields, there have been efforts to produce variable bioalcohol fuels, such as isobutanol and 2-phenylethanol, in order to meet industrial demands. YjgB is an aldehyde dehydrogenase from Escherichia coli that shows nicotinamide adenine dinucleotide phosphate (NADP)-dependent broad selectivity for aldehyde derivatives with an aromatic ring or small aliphatic chain. This could contribute to the design of industrial synthetic pathways. We determined the crystal structures of YjgB for both its apo-form and NADP-complexed form at resolutions of 1.55 and 2.00 Å, respectively, in order to understand the mechanism of broad substrate selectivity. The hydrophobic pocket of the active site and the nicotinamide ring of NADP(H) are both involved in conferring its broad specificity toward aldehyde substrates. In addition, based on docking-simulation data, we inferred that π-π stacking between substrates and aromatic side chains might play a crucial role in recognizing substrates. Our structural analysis of YjgB might provide insights into establishing frameworks to understand its broad substrate specificity and develop engineered enzymes for industrial biofuel synthesis.
Asunto(s)
Alcohol Deshidrogenasa/ultraestructura , Oxidorreductasas de Alcohol/ultraestructura , Proteínas de Escherichia coli/ultraestructura , Escherichia coli/enzimología , Conformación Proteica , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/genética , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Sitios de Unión/genética , Dominio Catalítico/genética , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ingeniería Metabólica , Modelos Moleculares , Especificidad por SustratoRESUMEN
The hPrp19-CDC5L complex plays a crucial role during human pre-mRNA splicing by catalytic activation of the spliceosome. In order to elucidate the molecular architecture of the hPrp19-CDC5L complex, the crystal structure of CTNNBL1, one of the major components of this complex, was determined. Unlike canonical ARM-repeat proteins such as ß-catenin and importin-α, CTNNBL1 was found to contain a twisted and extended ARM-repeat structure at the C-terminal domain and, more importantly, the protein formed a stable dimer. A highly negatively charged patch formed in the N-terminal ARM-repeat domain of CTNNBL1 provides a binding site for CDC5L, a binding partner of the protein in the hPrp19-CDC5L complex, and these two proteins form a complex with a stoichiometry of 2:2. These findings not only present the crystal structure of a novel ARM-repeat protein, CTNNBL1, but also provide insights into the detailed molecular architecture of the hPrp19-CDC5L complex.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Proteínas de Ciclo Celular/química , Enzimas Reparadoras del ADN/química , Glicoproteínas de Membrana/química , Proteínas Nucleares/química , Proteínas de Unión al ARN/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas del Dominio Armadillo/química , Proteínas del Dominio Armadillo/genética , Proteínas de Ciclo Celular/genética , Enzimas Reparadoras del ADN/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/genética , Proteínas Nucleares/genética , Unión Proteica/genética , Multimerización de Proteína/genética , Precursores del ARN/química , Precursores del ARN/genética , Empalme del ARN/genética , Factores de Empalme de ARN , Proteínas de Unión al ARN/genética , Secuencias Repetidas en TándemRESUMEN
ReBktB is a ß-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of ß-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K(m), V(max), and K(cat) values of 11.58 µM, 1.5 µmol/min, and 102.18 s(-1), respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.
Asunto(s)
Acetil-CoA C-Aciltransferasa/metabolismo , Cupriavidus necator/enzimología , Polihidroxialcanoatos/biosíntesis , Acetil-CoA C-Aciltransferasa/química , Acetil-CoA C-Aciltransferasa/genética , Secuencia de Aminoácidos , Biocatálisis , Cristalografía por Rayos X , Cupriavidus necator/metabolismo , Cinética , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Coronaviruses rely on host proteases to activate the viral spike protein, which facilitates fusion with the host cell membrane and the release of viral genomic RNAs into the host cell cytoplasm. The distribution of specific host proteases in the host determines the host, tissue, and cellular tropism of these viruses. Here, we identified the kallikrein (KLK) family member KLK5 as a major host protease secreted by human airway cells and exploited by multiple human betacoronaviruses. KLK5 cleaved both the priming (S1/S2) and activation (S2') sites of spike proteins from various human betacoronaviruses in vitro. In contrast, KLK12 and KLK13 displayed preferences for either the S2' or S1/S2 site, respectively. Whereas KLK12 and KLK13 worked in concert to activate SARS-CoV-2 and MERS-CoV spike proteins, KLK5 by itself efficiently activated spike proteins from several human betacoronaviruses, including SARS-CoV-2. Infection of differentiated human bronchial epithelial cells (HBECs) with human betacoronaviruses induced an increase in KLK5 that promoted virus replication. Furthermore, ursolic acid and other related plant-derived triterpenoids that inhibit KLK5 effectively suppressed the replication of SARS-CoV, MERS-CoV, and SARS-CoV-2 in HBECs and mitigated lung inflammation in mice infected with MERS-CoV or SARS-CoV-2. We propose that KLK5 is a pancoronavirus host factor and a promising therapeutic target for current and future coronavirus-induced diseases.
Asunto(s)
Calicreínas , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Replicación Viral , Humanos , Calicreínas/metabolismo , Calicreínas/genética , Animales , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Ratones , SARS-CoV-2/metabolismo , Betacoronavirus/metabolismo , Betacoronavirus/fisiología , COVID-19/metabolismo , COVID-19/virología , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Células HEK293 , Pulmón/virología , Pulmón/metabolismoRESUMEN
During the 2021/2022 winter season, we isolated highly pathogenic avian influenza (HPAI) H5N1 viruses harbouring an amino acid substitution from Asparagine(N) to Aspartic acid (D) at residue 193 of the hemagglutinin (HA) receptor binding domain (RBD) from migratory birds in South Korea. Herein, we investigated the characteristics of the N193D HA-RBD substitution in the A/CommonTeal/Korea/W811/2021[CT/W811] virus by using recombinant viruses engineered via reverse genetics (RG). A receptor affinity assay revealed that the N193D HA-RBD substitution in CT/W811 increases α2,6 sialic acid receptor binding affinity. The rCT/W811-HA193N virus caused rapid lethality with high virus titres in chickens compared with the rCT/W811-HA193D virus, while the rCT/W811-HA193D virus exhibited enhanced virulence in mammalian hosts with multiple tissue tropism. Surprisingly, a ferret-to-ferret transmission assay revealed that rCT/W811-HA193D virus replicates well in the respiratory tract, at a rate about 10 times higher than that of rCT/W811-HA193N, and all rCT/W811-HA193D direct contact ferrets were seroconverted at 10 days post-contact. Further, competition transmission assay of the two viruses revealed that rCT/W811-HA193D has enhanced growth kinetics compared with the rCT/W811-HA193N, eventually becoming the dominant strain in nasal turbinates. Further, rCT/W811-HA193D exhibits high infectivity in primary human bronchial epithelial (HBE) cells, suggesting the potential for human infection. Taken together, the HA-193D containing HPAI H5N1 virus from migratory birds showed enhanced virulence in mammalian hosts, but not in avian hosts, with multi-organ replication and ferret-to-ferret transmission. Thus, this suggests that HA-193D change increases the probability of HPAI H5N1 infection and transmission in humans.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Aviar , Animales , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Hemaglutininas , Virulencia , Hurones , PollosRESUMEN
Methylorubrum extorquens, a facultative methylotroph, assimilates C1 compounds and accumulates poly-ß-hydroxylbutyrate (PHB) as carbon and energy sources. The ethylmalonyl pathway is central to the carbon metabolism of M. extorquens, and is linked with a serine cycle and a PHB biosynthesis pathway. Understanding the ethylmalonyl pathway is vital in utilizing methylotrophs to produce value-added chemicals. In this study, we determined the crystal structure of the mesaconyl-CoA hydratase from M. extorquens (MeMeaC) that catalyzes the reversible conversion of mesaconyl-CoA to ß-methylmalyl-CoA. The crystal structure of MeMeaC revealed that the enzyme belongs to the MaoC-like dehydratase domain superfamily and functions as a trimer. In our current MeMeaC structure, malic acid occupied the substrate binding site, which reveals how MeMeaC recognizes the ß-methylmalyl-moiety of its substrate. The active site of the enzyme was further speculated by comparing its structure with those of other MaoC-like hydratases.
Asunto(s)
Acilcoenzima A , Carbono , Acilcoenzima A/metabolismo , Carbono/metabolismo , Vías BiosintéticasRESUMEN
The platform chemical 3-hydroxypropionic acid is used to synthesize various valuable materials, including bioplastics. Bifunctional malonyl-CoA reductase is a key enzyme in 3-hydroxypropionic acid biosynthesis as it catalyzes the two-step reduction of malonyl-CoA to malonate semialdehyde to 3-hydroxypropionic acid. Here, we report the cryo-EM structure of a full-length malonyl-CoA reductase protein from Chloroflexus aurantiacus (CaMCRFull). The EM model of CaMCRFull reveals a tandem helix architecture comprising an N-terminal (CaMCRND) and a C-terminal (CaMCRCD) domain. The CaMCRFull model also revealed that the enzyme undergoes a dynamic domain movement between CaMCRND and CaMCRCD due to the presence of a flexible linker between these two domains. Increasing the flexibility and extension of the linker resulted in a twofold increase in enzyme activity, indicating that for CaMCR, domain movement is crucial for high enzyme activity. We also describe the structural features of CaMCRND and CaMCRCD. This study reveals the protein structures underlying the molecular mechanism of CaMCRFull and thereby provides valuable information for future enzyme engineering to improve the productivity of 3-hydroxypropionic acid.
Asunto(s)
Oxidorreductasas , Microscopía por Crioelectrón , Oxidorreductasas/metabolismoRESUMEN
Glucuronic acid dehydrogenase (GluUADH), the product of the Csal-2474 gene from the halophilic bacterium Chromohalobacter salexigens DSM 3043, is an enzyme with potential use in the conversion of glucuronic acid in seaweed biomass to fuels and chemicals. GluUADH is an enzyme that catalyzes the oxidation of glucuronic acid (GluUA) and galacturonic acid (GalUA) and has a preference for NAD(+) rather than NADP(+) as a cofactor. Recombinant GluUADH was crystallized in the presence of 0.2â M calcium acetate, 0.1â M Tris-HCl pH 7.0 and 20% PEG 3000 at 295â K. X-ray diffraction data were collected to a maximum resolution of 2.1â Å. The GluUADH crystal belonged to space group P6(3), with unit-cell parameters a = b = 122.58, c = 150.49â Å, γ = 120°. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V(M)) is 2.78â Å(3)â Da(-1). The structure was solved by the single anomalous dispersion method and structure refinement is in progress.
Asunto(s)
Chromohalobacter/enzimología , Oxidorreductasas/química , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Expresión Génica , Oxidorreductasas/genética , Oxidorreductasas/aislamiento & purificaciónRESUMEN
SSADH is involved in the final step of GABA degradation, converting SSA to succinic acid in the human mitochondrial matrix, and its activity is known to be regulated via 'redox-switch modulation' of the catalytic loop. We present the crystal structure of EcSSADH, revealing that the catalytic loop of EcSSADH, unlike that of human SSADH, does not undergo disulfide bond-mediated structural changes upon changes of environmental redox status. Subsequent redox change experiments using recombinant proteins confirm the non-redox regulation of this protein. Detailed structural analysis shows that a difference in the conformation of the connecting loop (beta15-beta16) causes the formation of a water molecule-mediated hydrogen bond network between the connecting loop and the catalytic loop in EcSSADH, making the catalytic loop of EcSSADH more rigid compared to that of human SSADH. The cytosolic localization of EcSSADH and the cellular function of the GABA shunt in E. coli might result in the non-redox mediated regulatory mechanisms of the protein.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Succionato-Semialdehído Deshidrogenasa/química , Secuencia de Aminoácidos , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Citosol/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Estructura Secundaria de Proteína , Succionato-Semialdehído Deshidrogenasa/genéticaRESUMEN
Recovery of acetic acid (HAc) from the waste etching solution discharged from silicon wafer manufacturing process has been attempted by using solvent extraction process. For this purpose 2-ethylhexyl alcohol (EHA) was used as organic solvent. In the pre-treatment stage >99% silicon and hydrofluoric acid was removed from the solution by precipitation. The synthesized product, Na(2)SiF(6) having 98.2% purity was considered of commercial grade having good market value. The waste solution containing 279 g/L acetic acid, 513 g/L nitric acid, 0.9 g/L hydrofluoric acid and 0.030 g/L silicon was used for solvent extraction study. From the batch test results equilibrium conditions for HAc recovery were optimized and found to be 4 stages of extraction at an organic:aqueous (O:A) ratio of 3, 4 stages of scrubbing and 4 stages of stripping at an O:A ratio of 1. Deionized water (DW) was used as stripping agent to elute HAc from organic phase. In the whole batch process 96.3% acetic acid recovery was achieved. Continuous operations were successfully conducted for 100 h using a mixer-settler to examine the feasibility of the extraction system for its possible commercial application. Finally, a complete process flowsheet with material balance for the separation and recovery of HAc has been proposed.
Asunto(s)
Ácido Acético/aislamiento & purificación , Ácidos/química , Semiconductores , Solventes/química , Difracción de Rayos XRESUMEN
Microrchidia 3 (MORC3), a human ATPase linked to several autoimmune disorders, has been characterized both as a negative and positive regulator of influenza A virus. Here, we report that the CW domain of MORC3 (MORC3-CW) is targeted by the C-terminal tail of the influenza H3N2 protein NS1. The crystal structure of the MORC3-CW:NS1 complex shows that NS1 occupies the same binding site in CW that is normally occupied by histone H3, a physiological ligand of MORC3-CW. Comparable binding affinities of MORC3-CW to H3 and NS1 peptides and to the adjacent catalytic ATPase domain suggest that the viral protein can compete with the host histone for the association with CW, releasing MORC3 autoinhibition and activating the catalytic function of MORC3. Our structural, biochemical, and cellular analyses suggest that MORC3 might affect the infectivity of influenza virus and therefore has a role in cell immune response.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Unión al ADN/química , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Gripe Humana/metabolismo , Dominios Proteicos , Proteínas no Estructurales Virales/química , Adenosina Trifosfatasas/metabolismo , Unión Competitiva , Cristalografía por Rayos X , Proteínas de Unión al ADN/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Subtipo H3N2 del Virus de la Influenza A/fisiología , Gripe Humana/virología , Modelos Moleculares , Unión Proteica , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Histone methyltransferase MLL4 is centrally involved in transcriptional regulation and is often mutated in human diseases, including cancer and developmental disorders. MLL4 contains a catalytic SET domain that mono-methylates histone H3K4 and seven PHD fingers of unclear function. Here, we identify the PHD6 finger of MLL4 (MLL4-PHD6) as a selective reader of the epigenetic modification H4K16ac. The solution NMR structure of MLL4-PHD6 in complex with a H4K16ac peptide along with binding and mutational analyses reveal unique mechanistic features underlying recognition of H4K16ac. Genomic studies show that one third of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions in vivo and that MLL4 occupancy in a set of genomic targets depends on the acetyltransferase activity of MOF, a H4K16ac-specific acetyltransferase. The recognition of H4K16ac is conserved in the PHD7 finger of paralogous MLL3. Together, our findings reveal a previously uncharacterized acetyllysine reader and suggest that selective targeting of H4K16ac by MLL4 provides a direct functional link between MLL4, MOF and H4K16 acetylation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Dedos de Zinc PHD/fisiología , Acetilación , Animales , Sitios de Unión , Cromatina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Técnicas de Inactivación de Genes , Células HEK293 , Histona Acetiltransferasas/genética , N-Metiltransferasa de Histona-Lisina/química , Histonas/química , Humanos , Ratones Transgénicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Human p300 is a transcriptional co-activator and a major acetyltransferase that acetylates histones and other proteins facilitating gene transcription. The activity of p300 relies on the fine-tuned interactome that involves a dozen p300 domains and hundreds of binding partners and links p300 to a wide range of vital signaling events. Here, we report a novel function of the ZZ-type zinc finger (ZZ) of p300 as a reader of histone H3. We show that the ZZ domain and acetyllysine-recognizing bromodomain of p300 play critical roles in modulating p300 enzymatic activity and its association with chromatin. The acetyllysine binding function of bromodomain is essential for acetylation of histones H3 and H4, whereas interaction of the ZZ domain with H3 promotes selective acetylation of the histone H3K27 and H3K18 sites.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Dominios Proteicos , Dedos de Zinc , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Línea Celular , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Fluorescencia , Factores de Transcripción p300-CBP/químicaRESUMEN
Autophagic receptor p62 is a critical mediator of cell detoxification, stress response, and metabolic programs and is commonly deregulated in human diseases. The diverse functions of p62 arise from its ability to interact with a large set of ligands, such as arginylated (Nt-R) substrates. Here, we describe the structural mechanism for selective recognition of Nt-R by the ZZ domain of p62 (p62ZZ). We show that binding of p62ZZ to Nt-R substrates stimulates p62 aggregation and macroautophagy and is required for autophagic targeting of p62. p62 is essential for mTORC1 activation in response to arginine, but it is not a direct sensor of free arginine in the mTORC1 pathway. We identified a regulatory linker (RL) region in p62 that binds p62ZZ in vitro and may modulate p62 function. Our findings shed new light on the mechanistic and functional significance of the major cytosolic adaptor protein p62 in two fundamental signaling pathways.
Asunto(s)
Autofagia/fisiología , Proteína Sequestosoma-1/metabolismo , Autofagia/genética , Línea Celular , Cristalografía por Rayos X , Citometría de Flujo , Células HEK293 , Humanos , Inmunohistoquímica , Espectroscopía de Resonancia Magnética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Unión Proteica , Proteína Sequestosoma-1/genética , Transducción de Señal , Espectrometría de FluorescenciaAsunto(s)
Proteínas Bacterianas/química , Deshidrogenasas de Carbohidratos/química , Chromohalobacter/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dominio Catalítico , Coenzimas/química , Secuencia Conservada , Cristalografía por Rayos X , Ácido Glucurónico/química , Modelos Moleculares , Datos de Secuencia Molecular , NAD/química , Unión Proteica , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
The monocytic leukemia zinc-finger protein-related factor (MORF) is a transcriptional coactivator and a catalytic subunit of the lysine acetyltransferase complex implicated in cancer and developmental diseases. We have previously shown that the double plant homeodomain finger (DPF) of MORF is capable of binding to acetylated histone H3. Here we demonstrate that the DPF of MORF recognizes many newly identified acylation marks. The mass spectrometry study provides comprehensive analysis of H3K14 acylation states in vitro and in vivo. The crystal structure of the MORF DPF-H3K14butyryl complex offers insight into the selectivity of this reader toward lipophilic acyllysine substrates. Together, our findings support the mechanism by which the acetyltransferase MORF promotes spreading of histone acylation.
Asunto(s)
Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Histonas/química , Histonas/metabolismo , Acetilación , Sitios de Unión , Cristalografía por Rayos X , Células HeLa , Humanos , Lisina/química , Espectrometría de Masas , Unión Proteica , Dominios Proteicos , Procesamiento Proteico-PostraduccionalRESUMEN
Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome.