RESUMEN
Abasic sites are one of the most common DNA lesions. All known abasic site repair mechanisms operate only when the damage is in double-stranded DNA. Here, we report the discovery of 5-hydroxymethylcytosine (5hmC) binding, ESC-specific (HMCES) as a sensor of abasic sites in single-stranded DNA. HMCES acts at replication forks, binds PCNA and single-stranded DNA, and generates a DNA-protein crosslink to shield abasic sites from error-prone processing. This unusual HMCES DNA-protein crosslink intermediate is resolved by proteasome-mediated degradation. Acting as a suicide enzyme, HMCES prevents translesion DNA synthesis and the action of endonucleases that would otherwise generate mutations and double-strand breaks. HMCES is evolutionarily conserved in all domains of life, and its biochemical properties are shared with its E. coli ortholog. Thus, HMCES is an ancient DNA lesion recognition protein that preserves genome integrity by promoting error-free repair of abasic sites in single-stranded DNA.
Asunto(s)
5-Metilcitosina/análogos & derivados , Reparación del ADN/fisiología , ADN de Cadena Simple/fisiología , 5-Metilcitosina/metabolismo , Ácido Apurínico/metabolismo , ADN/metabolismo , Daño del ADN/fisiología , Replicación del ADN/fisiología , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas , Escherichia coli/metabolismo , Polinucleótidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
Osteoarthritis (OA) is characterized by degeneration of the joint cartilage, inflammation, and a change in the chondrocyte phenotype. Inflammation also promotes cell hypertrophy in human articular chondrocytes (HC-a) by activating the NF-κB pathway. Chondrocyte hypertrophy and inflammation promote extracellular matrix degradation (ECM). Chondrocytes depend on Smad signaling to control and regulate cell hypertrophy as well as to maintain the ECM. The involvement of these two pathways is crucial for preserving the homeostasis of articular cartilage. In recent years, Polynucleotides Highly Purified Technology (PN-HPT) has emerged as a promising area of research for the treatment of OA. PN-HPT involves the use of polynucleotide-based agents with controlled natural origins and high purification levels. In this study, we focused on evaluating the efficacy of a specific polynucleotide sodium agent, known as CONJURAN, which is derived from fish sperm. Polynucleotides (PN), which are physiologically present in the matrix and function as water-soluble nucleic acids with a gel-like property, have been used to treat patients with OA. However, the specific mechanisms underlying the effect remain unclear. Therefore, we investigated the effect of PN in an OA cell model in which HC-a cells were stimulated with interleukin-1ß (IL-1ß) with or without PN treatment. The CCK-8 assay was used to assess the cytotoxic effects of PN. Furthermore, the enzyme-linked immunosorbent assay was utilized to detect MMP13 levels, and the nitric oxide assay was utilized to determine the effect of PN on inflammation. The anti-inflammatory effects of PN and related mechanisms were investigated using quantitative PCR, Western blot analysis, and immunofluorescence to examine and analyze relative markers. PN inhibited IL-1ß induced destruction of genes and proteins by downregulating the expression of MMP3, MMP13, iNOS, and COX-2 while increasing the expression of aggrecan (ACAN) and collagen II (COL2A1). This study demonstrates, for the first time, that PN exerted anti-inflammatory effects by partially inhibiting the NF-κB pathway and increasing the Smad2/3 pathway. Based on our findings, PN can potentially serve as a treatment for OA.
Asunto(s)
FN-kappa B , Osteoartritis , Animales , Humanos , Masculino , FN-kappa B/metabolismo , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Polinucleótidos/farmacología , Polinucleótidos/metabolismo , Polinucleótidos/uso terapéutico , Células Cultivadas , Semen/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Osteoartritis/metabolismo , Condrocitos/metabolismo , Antiinflamatorios/farmacología , Hipertrofia/metabolismo , Interleucina-1beta/metabolismoRESUMEN
Early signalling events in response to elicitation include reversible protein phosphorylation and re-localization of plasma membrane (PM) proteins. Oligogalacturonides (OGs) are a class of damage-associated molecular patterns (DAMPs) that act as endogenous signals to activate the plant immune response. Previous data on early phosphoproteome changes in Arabidopsis thaliana upon OG perception uncovered the immune-related phospho-regulation of several membrane proteins, among which PCaP1, a PM-anchored protein with actin filament-severing activity, was chosen for its potential involvement in OG- and flagellin-triggered responses. Here, we demonstrate that PCaP1 is required for late, but not early, responses induced by OGs and flagellin. Moreover, pcap1 mutants, unlike the wild type, are impaired in the recovery of full responsiveness to a second treatment with OGs performed 24 h after the first one. Localization studies on PCaP1 upon OG treatment in plants expressing a functional PCaP1-GFP fusion under the control of PCaP1 promoter revealed fluorescence on the PM, organized in densely packed punctate structures, previously reported as microdomains. Fluorescence was found to be associated also with endocytic vesicles, the number of which rapidly increased after OG treatment, suggesting both an endocytic turnover of PCaP1 for maintaining its homeostasis at the PM and an OG-induced endocytosis.
Asunto(s)
Alarminas/metabolismo , Proteínas de Arabidopsis/fisiología , Arabidopsis/inmunología , Proteínas de Unión al Calcio/fisiología , Membrana Celular/metabolismo , Flagelina/metabolismo , Polinucleótidos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Botrytis , Proteínas de Unión al Calcio/metabolismo , Regulación de la Expresión Génica de las Plantas , Glucanos/metabolismo , Microscopía Confocal , Fosfoproteínas/metabolismo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Especies Reactivas de Oxígeno/metabolismo , TranscriptomaRESUMEN
N6-methyladenosine (m6A) plays an important role in various biological processes. Identifying m6A site is a key step in exploring its biological functions. One of the biggest challenges in identifying m6A sites is how to extract features comprising rich categorical information to distinguish m6A and non-m6A sites. To address this challenge, we propose bidirectional dinucleotide and trinucleotide position-specific propensities, respectively, in this paper. Based on this, we propose two feature-encoding algorithms: Position-Specific Propensities and Pointwise Mutual Information (PSP-PMI) and Position-Specific Propensities and Pointwise Joint Mutual Information (PSP-PJMI). PSP-PMI is based on the bidirectional dinucleotide propensity and the pointwise mutual information, while PSP-PJMI is based on the bidirectional trinucleotide position-specific propensity and the proposed pointwise joint mutual information in this paper. We introduce parameters α and ß in PSP-PMI and PSP-PJMI, respectively, to represent the distance from the nucleotide to its forward or backward adjacent nucleotide or dinucleotide, so as to extract features containing local and global classification information. Finally, we propose the M6A-BiNP predictor based on PSP-PMI or PSP-PJMI and SVM classifier. The 10-fold cross-validation experimental results on the benchmark datasets of non-single-base resolution and single-base resolution demonstrate that PSP-PMI and PSP-PJMI can extract features with strong capabilities to identify m6A and non-m6A sites. The M6A-BiNP predictor based on our proposed feature encoding algorithm PSP-PJMI is better than the state-of-the-art predictors, and it is so far the best model to identify m6A and non-m6A sites.
Asunto(s)
Adenosina/análogos & derivados , Algoritmos , Biología Computacional/métodos , Polinucleótidos/química , Procesamiento Postranscripcional del ARN , ARN/química , Adenosina/análisis , Adenosina/química , Adenosina/metabolismo , Humanos , Polinucleótidos/metabolismo , ARN/metabolismo , Análisis de Secuencia de ARN/métodosRESUMEN
Life as we know it requires three basic types of polymers: polypeptide, polynucleotide, and polysaccharide. Here we evaluate both universal and idiosyncratic characteristics of these biopolymers. We incorporate this information into a model that explains much about their origins, selection, and early evolution. We observe that all three biopolymer types are pre-organized, conditionally self-complementary, chemically unstable in aqueous media yet persistent because of kinetic trapping, with chiral monomers and directional chains. All three biopolymers are synthesized by dehydration reactions that are catalyzed by molecular motors driven by hydrolysis of phosphorylated nucleosides. All three biopolymers can access specific states that protect against hydrolysis. These protected states are folded, using self-complementary interactions among recurrent folding elements within a given biopolymer, or assembled, in associations between the same or different biopolymer types. Self-association in a hydrolytic environment achieves self-preservation. Heterogeneous association achieves partner-preservation. These universal properties support a model in which life's polymers emerged simultaneously and co-evolved in a common hydrolytic milieu where molecular persistence depended on folding and assembly. We believe that an understanding of the structure, function, and origins of any given type of biopolymer requires the context of other biopolymers.
Asunto(s)
Biopolímeros/biosíntesis , Biopolímeros/metabolismo , Biopolímeros/fisiología , Animales , Catálisis , Humanos , Péptidos/metabolismo , Péptidos/fisiología , Polímeros , Polinucleótidos/biosíntesis , Polinucleótidos/metabolismo , Polisacáridos/biosíntesis , Polisacáridos/metabolismo , Polisacáridos/fisiología , Pliegue de Proteína , Pliegue del ARN/fisiologíaRESUMEN
Retinoic acid-inducible gene I (RIG-I)-like receptors are cytosolic pattern recognition receptors (PRRs) that detect non-self-RNA and activate downstream interferon (IFN) signaling. One of the RIG-I-like receptors, laboratory of genetics and physiology 2 (LGP2), was originally thought to be a negative feedback regulator in the RIG-I signaling pathway, but growing evidence indicates that LGP2 is one cofactor of melanoma differentiation-associated protein 5 (MDA5) in MDA5-mediated IFN signaling activation. Our previous work showed that MDA5 was the major PRR to sense hepatitis C virus (HCV) infection in hepatocytes, but the role of LGP2 in HCV infection-induced IFN signaling has not been elucidated. In this study, we reported that LGP2 was a positive regulator of HCV infection-induced IFN signaling. Knockout of LGP2 in hepatocytes significantly diminished IFN production in response to HCV infection, but not to HCV 3'untranslated region RNA transfection. Mechanistic studies showed that LGP2 exerted its function at a step upstream of MDA5 in the IFN signaling. HCV infection promoted the molecular interaction between LGP2 and MDA5, which, in turn, enhanced MDA5/HCV RNA association. Finally, we demonstrated that the ATPase activity of LGP2 was critical for assisting MDA5/HCV RNA interaction and activating IFN signaling during HCV infection. CONCLUSION: Our work demonstrated that LGP2 plays an essential role in activating IFN signaling against HCV infection by promoting MDA5 recognition of HCV pathogen-associated molecular patterns. (Hepatology 2017;65:1478-1491).
Asunto(s)
Hepatitis C/inmunología , Helicasa Inducida por Interferón IFIH1/metabolismo , Interferones/metabolismo , ARN Helicasas/fisiología , Regiones no Traducidas 3' , Células HEK293 , Humanos , Inmunidad Innata , Poli I-C , Polinucleótidos/metabolismo , Virus Sendai/inmunologíaRESUMEN
Symmetrical cyclodextrin-based 14-arm star polymers with poly(ethylene glycol) PEG branches were synthesized and characterized. Interactions of the star polymers with lipid bilayers were studied by the "black lipid membrane" technique in order to demonstrate the formation of monomolecular artificial channels. The conditions for the insertion are mainly based on dimensions and amphiphilic properties of the star polymers, in particular the molar mass of the water-soluble polymer branches. Translocation of single-strand DNA (ssDNA) through those synthetic nanopores was investigated, and the close dimension between the cross-section of ssDNA and the cyclodextrin cavity led to an energy barrier that slowed down the translocation process.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Ciclodextrinas/química , Polietilenglicoles/química , Polinucleótidos/metabolismo , Secuencia de Bases , Transporte Biológico , ADN/genética , ADN/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismoRESUMEN
The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Modelos Moleculares , Proteínas Represoras/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Dicroismo Circular , ADN/química , Dimerización , Eliminación de Gen , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Difracción de Neutrones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Polinucleótidos/química , Polinucleótidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Desplegamiento Proteico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
We present terminal deoxynucleotidyl transferase-catalyzed enzymatic polymerization (TcEP) for the template-free synthesis of high-molecular-weight, single-stranded DNA (ssDNA) and demonstrate that it proceeds by a living chain-growth polycondensation mechanism. We show that the molecular weight of the reaction products is nearly monodisperse, and can be manipulated by the feed ratio of nucleotide (monomer) to oligonucleotide (initiator), as typically observed for living polymerization reactions. Understanding the synthesis mechanism and the reaction kinetics enables the rational, template-free synthesis of ssDNA that can be used for a range of biomedical and nanotechnology applications.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Polinucleótidos/metabolismo , Catálisis , ADN de Cadena Simple/metabolismo , Cinética , Peso Molecular , Nanotecnología , Polimerizacion , Polinucleótidos/químicaRESUMEN
Microbial rhodopsins are remarkable for the diversity of their functional mechanisms based on the same protein scaffold. A class of rhodopsins from cryptophyte algae show close sequence homology with haloarchaeal rhodopsin proton pumps rather than with previously known channelrhodopsins from chlorophyte (green) algae. In particular, both aspartate residues that occupy the positions of the chromophore Schiff base proton acceptor and donor, a hallmark of rhodopsin proton pumps, are conserved in these cryptophyte proteins. We expressed the corresponding polynucleotides in human embryonic kidney (HEK293) cells and studied electrogenic properties of the encoded proteins with whole-cell patch-clamp recording. Despite their lack of residues characteristic of the chlorophyte cation channels, these proteins are cation-conducting channelrhodopsins that carry out light-gated passive transport of Na(+) and H(+). These findings show that channel function in rhodopsins has evolved via multiple routes.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Criptófitas , Rodopsinas Sensoriales/metabolismo , Secuencia de Aminoácidos , Proteínas de Transporte de Catión/genética , Cationes Monovalentes/metabolismo , Chlorophyta , Evolución Molecular , Células HEK293 , Humanos , Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Luz , Técnicas de Placa-Clamp , Polinucleótidos/genética , Polinucleótidos/metabolismo , Protones , Rodopsinas Sensoriales/genética , Sodio/metabolismoRESUMEN
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) promotes catalytic scission of a phosphodiester bond between the 3'-end of DNA and the hydroxyl group of a tyrosine residue, as well as cleaving off a variety of other 3'-terminal phosphate-linked DNA substituents. We have shown recently that Tdp1 can initiate an apurinic/apyrimidinic (AP) site repair pathway that is independent from the one mediated by AP endonuclease 1 (APE1). Until recently, there was no method available of tracking the AP-site cleaving activity of Tdp1 by real-time fluorescence assay. In the present study we demonstrate a highly specific real-time detection of the AP-site cleaving activity of Tdp1 which allows one to distinguish it from the activity of APE1 by using a short hairpin oligonucleotide with a 1,12-dodecanediol loop, a 5'-fluorophore, and a 3'-quencher. Specific phosphodiesterase activity of Tdp1, which is usually able to remove quencher from the 3'-end of DNA, was suppressed in our approach by introducing a noncleavable phosphate group mimic between the 3'-end and the quencher. As a nondigestible 3'-phosphate analogue, we have used a new uncharged tetramethyl phosphoryl guanidine (Tmg) group, which is resistant to 3'-phosphodiesterase cleavage.
Asunto(s)
Ácido Apurínico/metabolismo , Bioensayo/métodos , Oligonucleótidos/química , Hidrolasas Diéster Fosfóricas/metabolismo , Polinucleótidos/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Colorantes Fluorescentes/química , Cinética , Microscopía Fluorescente , Mutación , Oligonucleótidos/metabolismo , Hidrolasas Diéster Fosfóricas/análisis , Hidrolasas Diéster Fosfóricas/genética , Especificidad por SustratoRESUMEN
We report the synthesis of two new amphiphilic conjugates 1 and 2 based on naphthalene di- and monoimide chromophores and the investigation of their photophysical, self-assembly and DNA-binding properties. These conjugates showed aqueous good solubility and exhibited strong interactions with DNA and polynucleotides such as poly(dGâ dC)-poly(dGâ dC) and poly(dAâ dT)-poly(dAâ dT). The interaction of these conjugates with DNA was evaluated by photo- and biophysical techniques. These studies revealed that the conjugates interact with DNA through intercalation with association constants in the order of 5-8×10(4) M(-1) . Of these two conjugates, bolaamphiphile 1 exhibited a supramolecular assembly that formed vesicles with an approximate diameter of 220â nm in the aqueous medium at a critical aggregation concentration of 0.4â mM, which was confirmed by SEM and TEM. These vesicular structures showed a strong affinity for hydrophobic molecules such as Nile red through encapsulation. Uniquely, when exposed to DNA the vesicles disassembled, and therefore this transformation could be utilised for the encapsulation and release of hydrophobic molecules by employing DNA as a stimulus.
Asunto(s)
Colorantes/química , ADN/química , Naftalenos/química , Polinucleótidos/química , ADN/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Polinucleótidos/metabolismoRESUMEN
BACKGROUND: Immunoglobulin class-switch recombination (CSR) is crucial for the expression of IgA, and it plays a vital role in the physiopathology of IgA nephropathy (IgAN). The aim of the study is to investigate the effect of polyriboinosinic:polyribocytidylic acid (poly(I:C)) in modulating toll-like receptor (TLR) 3-B-cell-activating factor belonging to the TNF family (BAFF) axis activation, which in turn promotes IgA CSR of IgAN patients and the IgAN rat model. METHODS: Blood samples and tonsillar tissue specimens were obtained from 24 patients with IgAN and 26 patients with chronic tonsillitis as control. We also used the IgAN rat model to investigate the relationship between viral infection and IgA CSR. RESULTS: Immunohistochemical and ELISA western blotting examination revealed that the TLR3/BAFF axis is activated in IgAN patients when compared to controls. Synthetic double-stranded RNA poly(I:C) stimulation upregulates the TACI/TLR3/TRIF/TRAF6 expression and promotes IgA CSR and BAFF productions in tonsil mononuclear cells. TLR3 or BAFF siRNA decreases IgA expression. In IgAN rat models, TLR3/BAFF signaling was highly activated. With 200 µg poly(I:C) sodium salt into the left naris for 8 weeks, IgA was highly deposited on glomeruli. It also revealed that poly(I:C) activated TLR3/BAFF axis and IgA CSR in vivo. CONCLUSION: These data points toward the role of TLR3/BAFF axis in IgA CSR of IgAN, and the data also support the notion that mucosal immunization with virus infection results in impaired mucosal and systemic IgA responses.
Asunto(s)
Factor Activador de Células B/metabolismo , Glomerulonefritis por IGA/inmunología , Cambio de Clase de Inmunoglobulina , Polinucleótidos/metabolismo , Receptor Toll-Like 3/metabolismo , Adolescente , Adulto , Animales , Modelos Animales de Enfermedad , Femenino , Glomerulonefritis por IGA/metabolismo , Humanos , Inmunoglobulina A , Masculino , Persona de Mediana Edad , Tonsila Palatina/metabolismo , Poli I-C , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Adulto JovenRESUMEN
A cherished tenet of nucleic acid enzymology holds that synthesis of polynucleotide 3'-5' phosphodiesters proceeds via the attack of a 3'-OH on a high-energy 5' phosphoanhydride: either a nucleoside 5'-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A(5')pp(5')N--in the case of polynucleotide ligases. RtcB exemplifies a family of RNA ligases implicated in tRNA splicing and repair. Unlike classic ligases, RtcB seals broken RNAs with 3'-phosphate and 5'-OH ends. Here we show that RtcB executes a three-step ligation pathway entailing (i) reaction of His337 of the enzyme with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate; (ii) transfer of guanylate to a polynucleotide 3'-phosphate to form a polynucleotide-(3')pp(5')G intermediate; and (iii) attack of a 5'-OH on the -N(3')pp(5')G end to form the splice junction. RtcB is structurally sui generis, and its chemical mechanism is unique. The wide distribution of RtcB proteins in bacteria, archaea, and metazoa raises the prospect of an alternative enzymology based on covalently activated 3' ends.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/metabolismo , Polinucleótidos/metabolismo , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/genética , Secuencia de Bases , Sitios de Unión , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Histidina/genética , Histidina/metabolismo , Cinética , Datos de Secuencia Molecular , Mutación , Fosfatos/metabolismo , ARN Ligasa (ATP)/genética , ARN Ligasa (ATP)/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismoRESUMEN
Although lipoproteins of mycoplasmas are thought to play a crucial role in interactions with their hosts, very few have had their biochemical function defined. The gene encoding the lipoprotein MslA in Mycoplasma gallisepticum has recently been shown to be required for virulence, but the biochemical function of this gene is not known. Although this gene has no significant sequence similarity to any gene of known function, it is located within an operon in M. gallisepticum that contains a homolog of a gene previously shown to be a nonspecific exonuclease. We mutagenized both genes to facilitate expression in Escherichia coli and then examined the functions of the recombinant proteins. The capacity of MslA to bind polynucleotides was examined, and we found that the protein bound single- and double-stranded DNA, as well as single-stranded RNA, with a predicted binding site of greater than 1 nucleotide but less than or equal to 5 nucleotides in length. Recombinant MslA cleaved into two fragments in vitro, both of which were able to bind oligonucleotides. These findings suggest that the role of MslA may be to act in concert with the lipoprotein nuclease to generate nucleotides for transport into the mycoplasma cell, as the remaining genes in the operon are predicted to encode an ABC transporter.
Asunto(s)
Proteínas Portadoras/genética , Lipoproteínas/genética , Mycoplasma gallisepticum/genética , Mycoplasma gallisepticum/patogenicidad , Polinucleótidos/genética , Polinucleótidos/metabolismo , Factores de Virulencia/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Infecciones por Mycoplasma/genética , Infecciones por Mycoplasma/metabolismo , Infecciones por Mycoplasma/microbiología , Mycoplasma gallisepticum/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The chemical and biological features of two newly synthesized [PtCl2(L)(2-aminonaphthalene)] complexes (L is NH3 or 2-aminonaphthalene) were compared with those of two already reported enantiomeric complexes of formula [PtCl2(DABN)] [DABN is (R)-1,1'-binaphthyl-2,2'-diamine or (S)-1,1'-binaphthyl-2,2'-diamine]. Solution behavior, lipophilicity, cytotoxicity with regard to one colorectal (HCT116) and two ovarian (A2780 and A2780Cp8) human carcinoma cell lines, and in vitro DNA- and G-quadruplex-binding properties were evaluated. In particular, the cytotoxicity of [PtCl2(NH3)(2-aminonaphthalene)] was better than that of cisplatin for all cell lines, and rather resembled that of oxaliplatin. The solution behavior of the whole series of complexes and the absence of an evident relationship between lipophilicity and cytotoxicity seem to suggest that all these experimental parameters are probably smoothed out during the 3-day cytotoxicity experiments and do not strongly affect the half-maximal inhibitory concentrations. The results of electrophoretic studies indicate that different kinds of interaction with DNA can be involved in the mode of action of these complexes, with intercalation in double-stranded DNA and stacking on G-quadruplex DNA being strongly implicated in particular for [PtCl2(NH3)(2-aminonaphthalene)].
Asunto(s)
Aminas/química , Carcinógenos/química , Cisplatino/análogos & derivados , ADN Superhelicoidal/metabolismo , Compuestos Organoplatinos/metabolismo , Compuestos Organoplatinos/farmacología , Polinucleótidos/metabolismo , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Células HCT116 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Compuestos Organoplatinos/síntesis química , Compuestos Organoplatinos/química , SolucionesRESUMEN
Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring â¼60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA-protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA-protein cross-links and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by â¼4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA-protein cross-links and generate double strand breaks, the most deleterious form of DNA damage.
Asunto(s)
Daño del ADN , ADN/metabolismo , Nucleosomas/metabolismo , Proteínas/metabolismo , Ácido Apurínico/química , Ácido Apurínico/genética , Ácido Apurínico/metabolismo , Secuencia de Bases , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , ADN/química , ADN/genética , Reparación del ADN , Electroforesis en Gel de Poliacrilamida , Histonas/química , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Nucleosomas/genética , Polinucleótidos/química , Polinucleótidos/genética , Polinucleótidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genéticaRESUMEN
Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the parvovirus adeno-associated virus type 2 (AAV-2) but does not inhibit retroviruses. In contrast, APOBEC3G (A3G) can block retroviruses but has only limited effects on AAV-2 or LINE-1. What dictates this differential target specificity remains largely undefined. Here, we modeled the structure of A3A based on its homology with the C-terminal domain of A3G and further compared the sequence of human A3A to those of 11 nonhuman primate orthologues. We then used these data to perform a mutational analysis of A3A, examining its ability to restrict LINE-1, AAV-2, and foreign plasmid DNA and to edit a single-stranded DNA substrate. The results revealed an essential functional role for the predicted single-stranded DNA-docking groove located around the A3A catalytic site. Within this region, amino acid differences between A3A and A3G are predicted to affect the shape of the polynucleotide-binding groove. Correspondingly, transferring some of these A3A residues to A3G endows the latter protein with the ability to block LINE-1 and AAV-2. These results suggest that the target specificity of APOBEC3 family members is partly defined by structural features influencing their interaction with polynucleotide substrates.
Asunto(s)
Citidina Desaminasa/química , Citidina Desaminasa/metabolismo , ADN de Cadena Simple/fisiología , Dependovirus/fisiología , Elementos de Nucleótido Esparcido Largo/fisiología , Polinucleótidos/metabolismo , Proteínas/química , Proteínas/metabolismo , Desaminasa APOBEC-3G , Animales , Secuencia de Bases , Citidina Desaminasa/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Células HEK293 , Haplorrinos , Células HeLa , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Polinucleótidos/química , Proteínas/genética , Retroelementos/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Interaction between tRNA and other polynucleotides with cytochrome c was studied by visible spectroscopy, fluorescence spectroscopy and gel mobility shift assay in view of the recently reported important regulatory role of tRNA in cytochrome c mediated apoptotic pathway. Visible spectroscopy showed perturbation in the heme binding environment in cytochrome c with tRNA binding. Fluorescence titrations indicated that cytochrome c binds to different polynucleotides with differing affinities. A weak binding was observed with single stranded polyribonucleotides and polydeoxyribonuleotides and strong binding with tRNA and double stranded DNA as indicated by extent of fluorescence quenching and binding constants. Calculation of thermodynamic binding parameters from fluorescence titrations indicated that three molecules of cytochrome c bound with one tRNA molecule with binding constant of 1.9 × 10(6) M(-1). The perturbation of cytochrome c structure caused by the binding of tRNA could be affecting its role in mediating apoptosis.
Asunto(s)
Citocromos c/metabolismo , Polinucleótidos/metabolismo , ARN de Transferencia/metabolismo , Apoptosis/fisiología , Unión ProteicaRESUMEN
HMGB1, one of the most abundant nuclear proteins, has a strong binding affinity for cisplatin-modified DNA. It has been proposed that HMGB1 enhances the anticancer efficacy of cisplatin by shielding platinated DNA lesions from repair. Two cysteine residues in HMGB1 domain A form a reversible disulfide bond under mildly oxidizing conditions. The reduced domain A protein binds to a 25-bp DNA probe containing a central 1,2-d(GpG) intrastrand cross-link, the major platinum-DNA adduct, with a 10-fold greater binding affinity than the oxidized domain A. The binding affinities of singly and doubly mutated HMGB1 domain A, respectively deficient in one or both cysteine residues that form the disulfide bond, are unaffected by changes in external redox conditions. The redox-dependent nature of the binding of HMGB1 domain A to cisplatin-modified DNA suggests that formation of the intradomain disulfide bond induces a conformational change that disfavors binding to cisplatin-modified DNA. Hydroxyl radical footprinting analyses of wild-type domain A bound to platinated DNA under different redox conditions revealed identical cleavage patterns, implying that the asymmetric binding mode of the protein across from the platinated lesion is conserved irrespective of the redox state. The results of this study reveal that the cellular redox environment can influence the interaction of HMGB1 with the platinated DNA and suggest that the redox state of the A domain is a potential factor in regulating the role of the protein in modulating the activity of cisplatin as an anticancer drug.