首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 187 毫秒
1.
The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein‐binding surface of an immobilized four‐way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ‐PNA1 and 4WJ‐PNA3). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ's. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two‐state equilibrium between: (i) an open‐x state favored by absence of Mg2+, low salt, and protein binding, and (ii) a compact stacked‐x state favored by Mg2+. 4WJ‐PNA3, like J1, shifts readily from an open to stacked conformation in the presence of Mg+2, while 4WJ‐PNA1 does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ‐PNA3 with nearly identical affinity (KDs) and 4WJ‐PNA1 with two‐fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs.  相似文献   

2.
3.
4.
5.
High mobility group box (HMGB) proteins are abundant nonhistone proteins found in all eukaryotic nuclei and are capable of binding/bending DNA. The human HMGB1 is composed of two binding motifs, known as Boxes A and B, are L-shaped alpha-helix structures, followed by a random-coil acidic tail that consists of 30 Asp and Glu residues. This work aimed at evaluating the role of the acidic tail of human HMGB1 in protein stability and DNA interactions. For this purpose, we cloned, expressed and purified HMGB1 and its tailless form, HMGB1ΔC, in E. coli strain. Tryptophan fluorescence spectroscopy and circular dichroism (CD) experiments clearly showed an increase in protein stability promoted by the acidic tail under different conditions, such as the presence of the chemical denaturant guanidine hydrochloride (Gdn.HCl), high temperature and low pH. Folding intermediates found at low pH for both proteins were denatured only in the presence of chemical denaturant, thus showing a relatively high stability. The acidic tail did not alter the DNA-binding properties of the protein, although it enhanced the DNA bending capability from 76° (HMGB1ΔC) to 91° (HMGB1), as measured using the fluorescence resonance energy transfer technique. A model of DNA bending in vivo was proposed, which might help to explain the interaction of HMGB1 with DNA and other proteins, i.e., histones, and the role of that protein in chromatin remodeling.  相似文献   

6.
Recent studies suggest that some RNA-binding proteins facilitate the folding of non-cognate RNAs. Here, we report that bacteriophage MS2 coat protein (MS2 CP) bound and promoted the catalytic activity of Candida group I ribozyme. Cloning of the MS2-bound RNA segments showed that this protein primarily interacts with the P5ab-P5 structure. Ultraviolet cross-linking and the T1 footprinting assay further showed that MS2 binding stabilized tertiary interactions, including the conserved L9-P5 interaction, and led to a more compact core structure. This mechanism is similar to that of the yeast mitochondrial tyrosyl-tRNA synthetase on other group I introns, suggesting that different RNA-binding proteins may use common mechanisms to support RNA structures.  相似文献   

7.
8.
9.
10.
Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently   总被引:4,自引:0,他引:4  
We analyzed the DNA-binding and DNA-bending properties of recombinant HMGB1 proteins based on a rice HMGB1 cDNA. Electrophoretic mobility shift assay demonstrated that rice HMGB1 can bind synthetic four-way junction (4H) DNA and DNA minicircles efficiently but the binding to 4H can be completed out by HMGA and histone H1. Conformational changes were detected by circular dichroism analysis with 4H DNA bound to various concentrations of HMGB1 or its truncated forms. T4 ligase-mediated circularization assays with short DNA fragments of 123 bp showed that the protein is capable of increasing DNA flexibility. The 123-bp DNA formed closed circular monomers efficiently in its presence, similar to that in an earlier study on maize HMG. Additionally, our results show for the first time that the basic N-terminal domain enhances the affinity of the plant HMGB1 protein for 4H DNA, while the acidic C-terminal domain has the converse effects.  相似文献   

11.
12.
13.
Summary Heterogeneous nuclear RNP protein A1, one of the major proteins in hnRNP particle (precursor for mRNA), is known to be post-translationally arginine-methylatedin vivo on residues 193, 205, 217 and 224 within the RGG box, the motif postulated to be an RNA binding domain. Possible effect of NG-arginine methyl-modification in the interaction of protein A1 to nucleic acid was investigated. The recombinant hnRNP protein A1 wasin vitro methylated by the purified nuclear protein/histone-specific protein methylase I (S-adenosylmethionine:protein-arginine N-methyltransferase) stoichiometrically and the relative binding affinity of the methylated and the unmethylated protein A1 to nucleic acid was compared: Differences in their binding properties to ssDNA-cellulose, pI values and trypsin sensitivities in the presence and absence of MS2-RNA all indicate that the binding property of hnRNP protein A1 to single-stranded nucleic acid has been significantly reduced subsequent to the methylation. These results suggest that posttranslational methyl group insertion to the arginine residue reduces protein-RNA interaction, perhaps due to interference of H-bonding between guanidino nitrogen arginine and phosphate RNA.Abbreviations hnRNP heterogeneous ribonucleoprotein particle - AdoMet S-adenosyl-L-methionine - AdoHcy S-adenosyl-L-homocysteine - MBP myelin basic protein - HMG high mobility group - ss single stranded  相似文献   

14.
15.
The mechanism by which sequence non-specific DNA-binding proteins enhance DNA flexibility is studied by examining complexes of double-stranded DNA with the high mobility group type B proteins HMGB2 (Box A) and HMGB1 (Box A+B) using atomic force microscopy. DNA end-to-end distances and local DNA bend angle distributions are analyzed for protein complexes deposited on a mica surface. For HMGB2 (Box A) binding we find a mean induced DNA bend angle of 78°, with a standard error of 1.3° and a SD of 23°, while HMGB1 (Box A+B) binding gives a mean bend angle of 67°, with a standard error of 1.3° and a SD of 21°. These results are consistent with analysis of the observed global persistence length changes derived from end-to-end distance measurements, and with results of DNA-stretching experiments. The moderately broad distributions of bend angles induced by both proteins are inconsistent with either a static kink model, or a purely flexible hinge model for DNA distortion by protein binding. Therefore, the mechanism by which HMGB proteins enhance the flexibility of DNA must differ from that of the Escherichia coli HU protein, which in previous studies showed a flat angle distribution consistent with a flexible hinge model.  相似文献   

16.
Survival rates for patients with pulmonary hypertension (PH) remain low, and our understanding of the mechanisms involved are incomplete. Here we show in a mouse model of chronic hypoxia (CH)-induced PH that the nuclear protein and damage-associate molecular pattern molecule (DAMP) high mobility group box 1 (HMGB1) contributes to PH via a Toll-like receptor 4 (TLR4)-dependent mechanism. We demonstrate extranuclear HMGB1 in pulmonary vascular lesions and increased serum HMGB1 in patients with idiopathic pulmonary arterial hypertension. The increase in circulating HMGB1 correlated with mean pulmonary artery pressure. In mice, we similarly detected the translocation and release of HMGB1 after exposure to CH. HMGB1-neutralizing antibody attenuated the development of CH-induced PH, as assessed by measurement of right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling and endothelial activation and inflammation. Genetic deletion of the pattern recognition receptor TLR4, but not the receptor for advanced glycation end products, likewise attenuated CH-induced PH. Finally, daily treatment of mice with recombinant human HMGB1 exacerbated CH-induced PH in wild-type (WT) but not Tlr4−/− mice. These data demonstrate that HMGB1-mediated activation of TLR4 promotes experimental PH and identify HMGB1 and/or TLR4 as potential therapeutic targets for the treatment of PH.  相似文献   

17.
SNCA/α-synuclein and its rare mutations are considered as the culprit proteins in Parkinson disease (PD). Wild-type (WT) SNCA has been shown to impair macroautophagy in mammalian cells and in transgenic mice. In this study, we monitored the dynamic changes in autophagy process and confirmed that overexpression of both WT and SNCAA53T inhibits autophagy in PC12 cells in a time-dependent manner. Furthermore, we showed that SNCA binds to both cytosolic and nuclear high mobility group box 1 (HMGB1), impairs the cytosolic translocation of HMGB1, blocks HMGB1-BECN1 binding, and strengthens BECN1-BCL2 binding. Deregulation of these molecular events by SNCA overexpression leads to autophagy inhibition. Overexpression of BECN1 restores autophagy and promotes the clearance of SNCA. siRNA knockdown of Hmgb1 inhibits basal autophagy and abolishes the inhibitory effect of SNCA on autophagy while overexpression of HMGB1 restores autophagy. Corynoxine B, a natural autophagy inducer, restores the deficient cytosolic translocation of HMGB1 and autophagy in cells overexpressing SNCA, which may be attributed to its ability to block SNCA-HMGB1 interaction. Based on these findings, we propose that SNCA-induced impairment of autophagy occurs, in part, through HMGB1, which may provide a potential therapeutic target for PD.  相似文献   

18.
19.
20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号