DNA损伤致p53-Mdm2相互作用的动力学模型

最新实验结果表明，在受到各种辐射而引起DNA损伤后，在单体细胞和群体细胞情况下，细胞中的p53蛋白浓度表现为非衰减振荡和衰减振荡两种不同的动力学行为.通过研究p53-Mdm2负反馈回路的非线性动力学行为，分析了各种(特别是DNA损伤、p53和 Mdm2蛋白浓度三者之间)动力学关系，提出了一个能同时描述这两种不同动力学行为的非线性模型. 关键词： p53-Mdm2负反馈回路 非衰减振荡和衰减振荡 非线性动力学模型     

A unified model for studying DNA damage-induced p53–Mdm2 interaction

Exploring the nonlinear dynamics of the negative feedback loop composed of p53 and Mdm2 proteins, we propose a signal–response model to study the dynamical mechanism of the different oscillatory behaviors for the activities of p53 and Mdm2 proteins both in individual and populations of cells. It will be shown that the sustained and damped oscillatory dynamics could be described in a unified way when the dynamics of the damage-derived signal is properly introduced.     

Mdm2生成速率调控的p53-Mdm2振子的全局动力学和稳定性

肿瘤抑制蛋白p53的动力学在一定程度上可以决定DNA损伤后的细胞命运.p53的动力学行为与p53信号通路中p53-Mdm2振子模块密切相关.然而,p53的负调控子Mdm2的生成速率的增加使其在一些癌细胞中过表达.因此探讨Mdm2生成速率对p53动力学的影响有重要意义.同时,PDCD5作为p53的激活子也调控p53的表达.因此,本文针对PDCD5调控的p53-Mdm2振子模型,通过分岔分析获得了Mdm2生成速率所调控的p53的单稳态、振荡以及单稳态与振荡共存的动力学行为,且稳定性通过能量面进行了分析.此外,噪声强度对p53动力学的稳定性有重要的影响.因此,针对p53的振荡行为,探讨了噪声强度对势垒高度和周期的影响.本文所获得的结果对理解DNA损伤后的p53信号通路调控起到一定的指导作用.     

DNA损伤致p53-Mdm2相互作用的非线性动力学模型(英文)

p53-Mdm2相互作用在DNA损伤的细胞响应方面起着非常重要的作用。最新实验结果表明,在受到各种辐射损伤而引起DNA损伤后,细胞中的p53蛋白浓度在单体细胞和群体细胞情况下,表现为非衰减振荡和衰减振荡两种不同的动力学行为。通过研究p53 -Mdm2负反馈回路的非线性动力学,分析了各种（特别是DNA损伤,p53和Mdm2浓度三者之间的）动力学关系,提出了一个能同时描述这两种不同动力学行为的非线性模型。 Exploring the nonlinear dynamics of the negative feedback loop composed of p53 and Mdm2 proteins, we propose a signal-response model to study the dynamical mechanism of the different oscillatory behaviors for the activities of p53 and Mdm2 proteins both in individual and population of cells. It is shown that the sustained and damped oscillatory dynamics could be described in a unified way when the dynamics of damage-derived signal is properly introduced.     

Mathematical simulation of complex formation of protein molecules allowing for their domain structure

A physical model of the interactions between protein molecules has been presented and an analysis of their propensity to form complex biological complexes has been performed. The reactivities of proteins have been studied using electrostatics methods based on the example of the histone chaperone Nap1 and histones H2A and H2B. The capability of proteins to form stable biological complexes that allow for different segments of amino acid sequences has been analyzed. The ability of protein molecules to form compounds has been considered by calculating matrices of electrostatic potential energy of amino acid residues constituting the polypeptide chain. The method of block matrices has been used in the analysis of the ability of protein molecules to form complex biological compounds.     

Ellagic acid and human cancers: a systems pharmacology and docking study to identify principal hub genes and main mechanisms of action

Research on anticancer properties of natural compounds, as effective materials that are available while causing minimal side effects, is growing. Ellagic acid (EA) is a well-known polyphenolic compound, which has been found in both free and complex modes in several medicinal plants such as pomegranate, walnut, and berries. Although many articles have reported anticancer properties for this compound, its mechanism of action has not been fully elucidated. In this study, we used several online and offline bioinformatics tools and databases to identify the mechanism of action of EA on various types of human malignancies including bladder, blood, breast, cervical, colorectal, liver, pancreas, and prostate cancers. In this context, after identifying and extracting EA-affected human genes/proteins that have been reported in various references, we built the related gene networks and determined functional hub genes. In addition, docking was performed to recognize target proteins that react directly with EA and are in fact most affected by this compound. Our findings revealed that EA exerts its anticancer effects by influencing specific hub genes in various types of cancers. Moreover, different cellular signaling pathways are affected by this natural compound. Generally, it turned out that EA probably exerts most of its anticancer activities, through induction of apoptosis, as well as P53 and WNT signaling pathways, and also by affecting the expression of several hub genes such as CDKN1A, CDK4, CDK2, CDK6, TP53, JUN, CCNA2, MAPK14, CDK1, and CCNB1 and especially interactions with some related proteins including P53, CDK6, and MAPK14.

     

EPR Characterization of Mononuclear Dinitrosyl Iron Complex with Persulfide as a New Representative of Dinitrosyl Iron Complexes in Biological Systems: an Overview

Some recent data on the presence of mononuclear dinitrosyl iron complexes (M-DNIC) with persulfide (R-S-S^?) ligands with a characteristic electron paramagnetic resonance signal at g _⊥ = 2.35 and g _|| = 2.02 (g _aver. = 2.03) in biological systems (e.g., Escherichia coli cells and isolated iron–sulfur proteins) are reviewed. The generation of M-DNIC is controlled by inorganic sulfur (sulfide, S^2?) whose binding to thiols gives persulfides. It is suggested that enhanced production of inorganic sulfur is a result of destruction of active centers of iron–sulfur proteins in the presence of NO or NO-containing compounds. Dinitrosyl iron complexes with thiol-containing ligands are the most active participants in this process. Inorganic sulfur may appear in biological systems during the synthesis or resynthesis of active centers of iron–sulfur proteins in response to activation of cysteine desulfurase, the key enzyme in sulfide synthesis from cysteine.     

Microscopy techniques in flavivirus research

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus–host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses.     

Atomic force microscopy: determination of unbinding force, off rate and energy barrier for protein-ligand interaction

Recently, atomic force microscopy (AFM) based force measurements have been applied biophysically and clinically to the field of molecular recognition as well as to the evaluation of dynamic parameters for various interactions between proteins and ligands in their native environment. The aim of this review is to describe the use of the AFM to measure the forces that control biological interaction, focusing especially on protein-ligand and protein-protein interaction modes. We first considered the measurements of specific and non-specific unbinding forces which together control protein-ligand interactions. As such, we will look at the theoretical background of AFM force measurement curves for evaluating the unbinding forces of protein-ligand complexes. Three AFM model dynamic parameters developed recently for use in protein-ligand interactions are reviewed: (i) unbinding forces, (ii) off rates, and (iii) binding energies. By reviewing the several techniques developed for measuring forces between biological structures and intermolecular forces in the literature, we show that use of an AFM for these applications provides an excellent tool in terms of spatial resolution and lateral resolution, especially for protein-protein and protein-ligand interactions.     

Comparison of ligand migration and binding in heme proteins of the globin family

The binding of small diatomic ligands such as carbon monoxide or dioxygen to heme proteins is among the simplest biological processes known. Still, it has taken many decades to understand the mechanistic aspects of this process in full detail. Here, we compare ligand binding in three heme proteins of the globin family, myoglobin, a dimeric hemoglobin, and neuroglobin. The combination of structural, spectroscopic, and kinetic experiments over many years by many laboratories has revealed common properties of globins and a clear mechanistic picture of ligand binding at the molecular level. In addition to the ligand binding site at the heme iron, a primary ligand docking site exists that ensures efficient ligand binding to and release from the heme iron. Additional, secondary docking sites can greatly facilitate ligand escape after its dissociation from the heme. Although there is only indirect evidence at present, a preformed histidine gate appears to exist that allows ligand entry to and exit from the active site. The importance of these features can be assessed by studies involving modified proteins(via site-directed mutagenesis) and comparison with heme proteins not belonging to the globin family.     

Insight into binding modes of p53 and inhibitors to MDM2 based on molecular dynamic simulations and principal component analysis

Inhibition of the p53–MDM2 interaction is a new therapeutic strategy to activate the wild-type function of p53 in tumors. Molecular dynamics (MD) simulations and calculations of binding free energies were performed to investigate the binding mechanisms of p53 and two inhibitors PMI and VZV to MDM2. The results show that van der Waals interaction is the main force to control the bindings of ligands to the hydrophobic cleft of MDM2, which basically agrees with the previous calculated and experimental studies. The results from the RMSF calculation, cross-correlation analysis and principal component (PC) analysis prove that the ligand bindings produce a significant effect on the conformation of the binding cleft of MDM2. In addition, the calculations of residue-based free energy decomposition suggest that the CH–CH, CH–π, and π–π interactions dominate the bindings of p53 and inhibitors to MDM2. This study can provide significant help for the design of potent inhibitors targeting the p53–MDM2 interaction.     

Implications of glutathione-S transferase P1 in MAPK signaling as a CRAF chaperone: In memory of Dr. Irving Listowsky

Glutathione-S transferase P1 (GSTP1) is one of the glutathione-S transferase isozymes that belong to a family of phase II metabolic isozymes. The unique feature of GSTP1 compared with other GST isozymes is its relatively high expression in malignant tissues. Thus, clinically, GSTP1 serves as a tumor marker and as a refractory factor against certain types of anticancer drugs through its primary function as a detoxifying enzyme. Additionally, recent studies have identified a chaperone activity of GSTP1 involved in the regulation the function of various intracellular proteins, including factors of the growth signaling pathway. In this review, we will first describe the function of GSTP1 and then extend the details onto its role in the mitogen-activated protein kinase signal pathway, referring to the results of our recent study that proposed a novel autocrine signal loop formed by the CRAF/GSTP1 complex in mutated KRAS and BRAF cancers. Finally, the possibilities of new therapeutic approaches for these cancers by targeting this complex will be discussed.     

基于p53DNA的泽泻醇抗肿瘤分子机制研究

研究泽泻醇类化合物23-乙酰泽泻醇B(alisol B 23-acetate, 23B)、24-乙酰泽泻醇A(alisol A 24-acetate, 24A)混合物(24A∶23B含量比=1∶1)与抑癌基因p53DNA的作用机理,探讨泽泻醇类化合物抗肿瘤作用的分子机制。紫外-可见吸收光谱法、荧光光谱法与分子模拟联用探讨23B,24A及24A-23B混合物与p53DNA的作用方式。紫外光谱显示泽泻醇单体与其混合物部分嵌插入p53DNA,他们使p53DNA紫外吸收降低的程度为：24A∶23B(1∶1)>23B>24A。荧光光谱显示泽泻醇单体及其混合物与p53DNA的相互作用模式均为嵌插结合,结合强度为：24A∶23B(1∶1)>23B>24A。分子模拟显示,泽泻醇单体及其混合物与p53DNA结合能的大小顺序为：24A∶23B (1∶1)>23B>24A,23B与p53DNA f 链的腺嘌呤脱氧核苷酸(DA4)形成1个氢键,24A-23B复合物与p53DNA的DA4、胸腺嘧啶脱氧核苷酸(DT19)形成4个氢键。24A,23B及其混合物与p53DNA结合的强度顺序：24A∶23B (1∶1)>23B>24A,表明24A和23B对抗癌靶点p53DNA具有协同作用,三者与p53DNA的作用方式均为部分嵌插结合。同时,泽泻醇化合物母环C14-和结构中的空间位阻,p53DNA f 链的DA4中磷酸上的氧原子为泽泻醇类化合物与p53DNA相互作用的关键结合位点,是该类泽泻醇发挥抗肿瘤作用的活性中心。24A侧链C19-上的羟基,p53DNA f 链的DA4中腺嘌呤上的氮原子和氧原子,e链的DT19中胸腺嘧啶上的氧原子为泽泻醇类化合物协同增效作用的关键。     

Effect of 1-Aminoanthracene (1-AMA) Binding on the Structure of Three Lipocalin Proteins,the Dimeric β Lactoglobulin,the Dimeric Odorant Binding Protein and the Monomeric α<Subscript>1</Subscript>-Acid Glycoprotein. Fluorescence Spectra and Lifetimes Studies

We studied effect of 1-aminoanthracene (1-AMA) binding on the structures of dimeric β lactoglobulin, dimeric odorant binding protein (OBP) and monomeric α₁-acid glycoprotein (lipocalin family proteins) by monitoring fluorescence excitation spectra and measuring fluorescence lifetimes of the tryptophan residues of the proteins. Results show that binding of 1-AMA to β lactoglobulin and OBP modifies their conformation even at low probe concentration compared to that of the proteins. Structural modification induces a red shift of the fluorescence excitation spectra maximum of tryptophan residues accompanied with an increase of the third fluorescence lifetime and a decrease of its pre-exponential factor. These effects were not observed for α₁-acid glycoprotein, probably as the result of carbohydrate presence. These data raise doubts concerning use of 1-AMA as a probe to study biological properties of β lactoglobulin and OBP.     

丹酚酸B与牛血清白蛋白相互作用的光谱学研究

丹酚酸B（SAB）是丹参中主要的水溶性成分之一,具有广泛的生物活性。血清白蛋白是哺乳动物体内血浆中含量最为丰富的蛋白质,约占血浆总蛋白的60%,能与许多内源及外源性物质相结合,发挥存储和转运的作用。丹酚酸B进入人体后,必然先与血液中的蛋白质相结合,然后才被转运到其受体结合部位,进而发挥其药理作用。为更好地了解丹酚酸B在体内的分布、转运及代谢,在模拟生理条件下,采用荧光光谱法、圆二色光谱法和核磁共振波谱法等方法研究丹酚酸B与牛血清白蛋白（BSA）的相互作用机制。结果表明：丹酚酸B与牛血清白蛋白的结合能有效地导致牛血清白蛋白的内源荧光猝灭,猝灭机制为以静态猝灭为主的联合猝灭方式。荧光光谱分析表明两者的结合常数分别为7.51 ×105（288 K）,7.40 ×105（298 K）和5.57 ×105（308 K） L·mol -1,达到105 L·mol -1数量级,且随着温度的升高逐渐降低。Scatchard方法确定牛血清白蛋白与丹酚酸B相互作用时结合位点数约为1,说明两者之间可形成1∶1型非共价复合物。位点标记竞争实验表明丹酚酸B在牛血清白蛋白亚结构域IIA（Site I）的疏水腔内相结合。三维荧光光谱法和圆二色谱法实验结果显示,结合丹酚酸B后牛血清白蛋白中色氨酸和酪氨酸所处的微环境发生了一定的变化（即峰位置发生红移,色氨酸和酪氨酸残基周围微环境疏水性减小,极性增强）,而二级结构和和三级结构的变化较小。此外,利用核磁共振波谱技术比较一定浓度的丹酚酸B在不同浓度的牛血清白蛋白溶液中的化学位移变化情况,研究表明H5”和H6”所在的苯环在牛血清白蛋白与丹酚酸B相互作用过程中发挥着重要的作用。该研究有助于了解丹酚酸B在机体内的作用机制以及对血清白蛋白结构和功能的影响,为丹酚酸B类新药的研发提供一定的理论参考。     

Rhythm generation by the pre-Bötzinger Complex in medullary slice and island preparations: Effects of adenosine A1 receptor activation

Background

Alpha-Synuclein (α-syn), a 140 amino acid protein associated with presynaptic membranes in brain, is a major constituent of Lewy bodies in Parkinson's disease (PD). Three missense mutations (A30P, A53T and E46K) in the α-syn gene are associated with rare autosomal dominant forms of familial PD. However, the regulation of α-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood.

Results

In the present study, we analysed the ability of cytosolic factors to regulate α-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant α-syn. To characterize cytosolic factor(s) that modulate α-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate α-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T α-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance α-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P α-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.

Conclusion

These findings suggest that endogenous brain cytosolic factors regulate Wt and mutant α-syn membrane binding, and could represent potential targets to influence α-syn solubility in brain.     

Human immunodeficiency virus type 1 efficiently binds to human fetal astrocytes and induces neuroinflammatory responses independent of infection

Background  

HIV-1 infects human astrocytes in vitro and in vivo but the frequency of infected cells is low and its biological significance is unknown. In studies in vitro, recombinant gp120 alone can induce profound effects on astrocyte biology, suggesting that HIV-1 interaction with astrocytes and its functional consequences extend beyond the limited levels of infection in these cells. Here we determined the relative efficiencies of HIV-1 binding and infection in human fetal astrocytes (HFA), mainly at the single cell level, using HIV-1 tagged with green fluorescence protein (GFP)-Vpr fusion proteins, termed HIV-GFP, to detect virus binding and HIV-1 expressing Rev and NefGFP fusion proteins to detect productive infection.     

Production of biologically active peptides by hydrolysis of whey protein isolates using hydrodynamic cavitation

Whey protein isolate (WPI) hydrolysates have higher solubility in aqueous phase and enhanced biological properties. Hydrolysis of WPI was optimized using operating pressure (ΔP, bar), number of passes (N), and WPI concentration (C, %) as deciding parameters in hydrodynamic cavitation treatment. The optimum conditions for generation of WPI hydrolysate with full factorial design were 8 bar, 28 passes, and 4.5% WPI concentration yielding 32.69 ± 1.22 mg/mL soluble proteins. WPI hydrolysate showed alterations in binding capacity over WPI. SDS-PAGE and particle size analysis confirmed the hydrolysis of WPI. Spectroscopic, thermal and crystallinity analyses showed typical properties of proteins with slight variations after hydrodynamic cavitation treatment. ABTS, DPPH and FRAP assays of WPI hydrolysate showed 7–66, 9–149, and 0.038–0.272 µmol/mL GAE at 1–10, 0.25–4, and 3–30 mg/mL concentration, respectively. Further, a considerable enhancement in fresh weight, chlorophyll, carotenoids, reducing sugars, total soluble sugars, soluble proteins content and total phenolics content was noticed during in vitro growth of sugarcane in WPI hydrolysate supplemented medium at 50–200 mg/L concentration over the control. The process cost (INR/kg) to hydrolyze WPI was also calculated.