ZBP1-Mediated Necroptosis: Mechanisms and Therapeutic Implications

Cell death is a fundamental pathophysiological process in human disease. The discovery of necroptosis, a form of regulated necrosis that is induced by the activation of death receptors and formation of necrosome, represents a major breakthrough in the field of cell death in the past decade. Z-DNA-binding protein (ZBP1) is an interferon (IFN)-inducing protein, initially reported as a double-stranded DNA (dsDNA) sensor, which induces an innate inflammatory response. Recently, ZBP1 was identified as an important sensor of necroptosis during virus infection. It connects viral nucleic acid and receptor-interacting protein kinase 3 (RIPK3) via two domains and induces the formation of a necrosome. Recent studies have also reported that ZBP1 induces necroptosis in non-viral infections and mediates necrotic signal transduction by a unique mechanism. This review highlights the discovery of ZBP1 and its novel findings in necroptosis and provides an insight into its critical role in the crosstalk between different types of cell death, which may represent a new therapeutic option.     

含铋配合物与奈瑟氏菌铁结合蛋白的反应性质研究

铁结合蛋白(Fbp)是致病菌获取Fe3+的关键蛋白。本文采用氨三乙酸(H3NTA)和硝酸铋反应制备BiNTA.2H2O,并运用元素分析、NMR等手段进行表征。通过在大肠杆菌中克隆表达和分离纯化出奈瑟氏淋病双球菌的Fbp,测定不同计量比nBiNTA/napo-Fbp下反应的紫外可见光谱,确定BiNTA与apo-Fbp的反应为一级反应,反应速率常数约为(0.175±0.064)min-1(10 mmol.L-1 Hepes/HPO42-pH 7.4缓冲溶液,310 K)。NH4BiCit与apo-Fbp的反应也为一级反应,反应速率与BiNTA相近。Bi3+与apo-Fbp的饱和结合计量比为1∶1,形成三元配合物Fbp-Bi-NTA的结合常数(lgK)为(21.43±0.20),形成Fbp-Bi-Cit的结合常数(lgK)为(16.03±0.03)。实验结果表明,致病菌中运输Fe3+的蛋白铁结合蛋白可作为含铋抗菌药物的潜在靶分子。     

Studies of Adriamycin Binding to Histone H1 by Resonant Mirror Biosensor and Fluorescence Spectroscopy

Abstract

Adriamycin is a clinically used antitumor anthracycline antibiotic. Histone H1 is a target for the activity of anthracycline drug at the chromatin level. A new optical biosensor technique based on the resonant mirror was used to characterize interaction of adriamycin with H1, and the binding constant was obtained. By the analysis of fluorescence spectrum and fluorescence intensity, it was shown that adriamycin can quench the intrinsic fluorescence of tyrosine in H1 through a static quenching procedure. The binding constants of adriamycin with H1 were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained, and the binding forces were suggested to be mainly hydrophobic. The interaction of adriamycin and H1 in the presence of denaturant or salt was studied. The effect of Fe³⁺ on the binding constant was also investigated by optical biosensor and fluorescence spectroscopy. Furthermore, the steady‐state Stern–Volmer collisional quenching study of Tyr72 with acrylamide indicated that the association between adriamycin and H1 did not change molecular conformation of H1.     

Structure Analysis of Streptococcal Protein G Fc Binding Domain

The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein Gnucleotide sequences, and resultsin an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA.The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm.It shows a typical α-helix region in this domain.By breaking this α-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG.The hydropathicity of this domain was also analyzed and compared with that of protein A relevant     

毛细管区带电泳法测定4种抗HIV-1活性的化合物与牛血清白蛋白的结合常数

采用毛细管区带电泳紫外检测方法,在pH 8.0、浓度50 mmol/L的磷酸盐缓冲溶液及运行电压15 kV的条件下,测定了4种新合成的具有抗HIV-1活性的化合物(IG3,iso-C3,C3,MC3)与牛血清白蛋白(BSA)相互作用的结合常数。在缓冲溶液中加入不同浓度的BSA,通过测定化合物迁移时间的变化,计算得到了上述4种化合物与BSA的结合常数分别为1.07×104, 1.34×104, 8.51×103和9.45×103 L/mol。该方法简单、快捷,可用于研究结合比为1∶1的小分子与生物大分子的     

泛昔洛韦解离常数和结合常数的测定

采用紫外分光光度法,在225 nm与305 nm波长处测得泛昔洛韦的解离常数pKa均为3.8.在含有泛昔洛韦的pH值为7.0的Na2HPO4-KH2PO4缓冲溶液中加入牛血清白蛋白,以双倒数公式法测定二者的结合常数.结果表明,牛血清白蛋白与泛昔洛韦形成了1∶4的配合物,其结合常数是4.51×106 L/mol.方法简便,结果可靠。     

毛细管电泳测定主客分子相互作用的结合常数

在２５℃下,用毛细管区带电泳测定了主管体分子（配体和溶质分子）β－环糊精（β－ＣＤ）和心得舒（ａｌｐｒｅｎｏｌｏ）在ｐＨ值为２．５,浓度为１００ｍｍｏｌ／Ｌ的磷酸盐缓冲溶液下的结合常数,并对４种求解方法既非线性拟合法,双倒数法,ｙ－倒数法和ｘ－倒数法的结果进行了比较,所得值分别是３０７．２、４０８．４、３３１．４和３４３．１Ｌ／ｍｏｌ,同时获得了结合物的迁移率。该方法可用于测定结合比为１：１的各种     

Metal Ion Dependent Binding and Nuclease Activity of Hexaazacyclophane

The HAPP-Fe complex was more active than HAPP-Cu complex in cleavage of DNA in the presence of dithiothreitol and hydrogen peroxide. In contrast, the HAPP-Cu complex was found to bind more strongly with λ-DNA, whereas the HAPP-Fe binds weakly. It was shown that the metal ion complexed with HAPP ligand can influence the binding and cleaving activity due to the ability of the ligand to undergo geometrical tautomerization.     

Isomeric Effect on the anticancer Behavior of two Zinc (II) complexes based on 3,5‐bis(1‐imidazoly) pyridine: Experimental and Theoretical Approach

Two isomeric Zinc (II) complexes constructed by 3,5‐bis(1‐imidazoly) pyridine has been synthesized and characterized by single crystal X‐ray diffraction, elemental analyses and infrared spectroscopy. The binding mode and ability of complex 1–2 with CT‐DNA were studied by UV and fluorescence spectra. The intrinsic binding constant K_b (K_b1 = 2.305 × 10⁴ M^?1, K_b2 = 3.095 × 10⁴ M^?1) and the observed association constant K_obs (K_obs1 = 1.523*10⁶ M^?1, K_obs2 = 2.057*10⁶ M^?1) indicated that the insertion ability of complex 2 with CT‐DNA is stronger than complex 1. Gel electrophoresis showed that complexes have a good ability to hydrolyze cleavage pBR322 plasmid DNA. The cytotoxicity and apoptosis studies showed that complexes exhibited excellent cytotoxic activity against HeLa cells, especially complex 2 had better growth inhibition than Cisplatin. Molecular docking study simulated the binding model of complexes with DNA (PDB:4av1), showing an imidazole plane of complex 2 can be inserted into a DNA base pair in relative parallel. Both complexes can be used as potential anticancer agents.     

Copper Coordination Polymers of N1,N4-Bis(pyridin-2-ylmethyl)terephthalamide: Synthesis,Structures, DNA Binding and Cleavage,and Catalytic Activity for Oxidation of 1-Phenylethanol

Two Cu coordination polymers [Cu(bpmt)Cl₂ · 2DMF]_n ( 1 ), [Cu(bpmt)(μ-Cl) · DMSO]_n ( 2 ) [bpmt = N¹,N⁴-bis(pyridin-2-ylmethyl)terephthalamide)], were synthesized and characterized. X-ray single-crystal diffractions revealed complex 1 exhibits a one-dimensional (1D) chain in which central Cu^II ions are located in a six-coordinate slightly distorted octahedral structure. Complex 2 features 1D chain composed of Cu^I–Cl bridges and all the central Cu^I ions are situated a twisted tetrahedral configurations. Multi-spectroscopic DNA interaction studies indicate that the two complexes may bind to DNA via groove binding modes, and the calculated binding constants are 1.42 × 10⁴ ( 1 -CT-DNA) and 1.41 × 10⁴ ( 2 -CT-DNA). Both complexes are also capable of efficiently cleaving plasmid pBR322 DNA in the presence of H₂O₂ via an oxidative mechanism. Furthermore, 1 and 2 are employed as catalysts for the oxidation of 1-phenylethanol to acetophenone. Complex 1 displays good catalytic potential with the conversion rate reaches 99 % using tert-butyl hydroperoxide (TBHP) as oxidant and CH₃CN as solvent at 60 °C for 2.0 h.     

Interaction of Cationic Manganese Porphyrins with DNA. A Binding Model

The DNA cleavage properties of two cationic manganese porphyrins possessing different peripheral substituents were compared. The identical nature of the strand scission patterns of the porphyrins on a 139 base pair restriction fragment of pBR-322 DNA, along with other evidence, suggests that the porphyrin is end-on bound via the minor groove in a melted or partially melted region of DNA. This unusual binding mode underscores the potential of outisde binding cationic metalloporphyrins as probes for low melting regions of DNA     

金属元素组与血清白蛋白的竞争结合反应性能研究

利用亲和毛细管电泳（Affinitycapillaryelectrophoresis,ACE）法研究金属元素组和血清白蛋白（Bovineserumalbumin,BSA）的竞争结合反应性能。基于位点结合模型,构建双金属组[Zn²⁺,Cu²⁺]与血清白蛋白结合反应模型,建立多元金属组与生物大分子竞争结合的理论方程,测定结合参数并解析动力学机制。结果表明,金属元素组[Zn²⁺,Cu²⁺]与BSA发生竞争结合反应形成配合物Zn²⁺-BSA和Cu²⁺-BSA。依据有效淌度变化,通过建立的理论方程非线性拟合竞争结合反应的平均表观结合常数K_Zn²⁺-BSA=4.01×10⁴L·mol^-1、K_Cu²⁺-BSA=7.75×10⁴L·mol^-1。结合反应均为快平衡反应,Cu²⁺对Zn²⁺离子的结合作用有明显拮抗作用。分析ACE谱显示配合物的峰高与配体结合能力大小、配合物稳定性之间存在量效关系。     

金属元素组与血清白蛋白的竞争结合反应性能研究

利用亲和毛细管电泳(Affinity capillary electrophoresis,ACE)法研究金属元素组和血清白蛋白(Bovine serum albumin,BSA)的竞争结合反应性能。基于位点结合模型,构建双金属组[Zn2+,Cu2+]与血清白蛋白结合反应模型,建立多元金属组与生物大分子竞争结合的理论方程,测定结合参数并解析动力学机制。结果表明,金属元素组[Zn2+,Cu2+]与BSA发生竞争结合反应形成配合物Zn2+-BSA和Cu2+-BSA。依据有效淌度变化,通过建立的理论方程非线性拟合竞争结合反应的平均表观结合常数KZn2+-BSA=4.01×104L·mol-1、KCu2+-BSA=7.75×104L·mol-1。结合反应均为快平衡反应,Cu2+对Zn2+离子的结合作用有明显拮抗作用。分析ACE谱显示配合物的峰高与配体结合能力大小、配合物稳定性之间存在量效关系。     

A Practical Guide for the Determination of Binding Constants

When working in the field ofhost–guest chemistry, the binding constants have to bedetermined on many occasions. Here is a detaileddocument of how to determine the binding constantswhich covers both the basic principle and thepractical issue: a practical experimental guideline,a representative method for the determination ofstoichiometry and for the evaluation of a complexconcentration, precautions to be taken on setting upconcentration conditions of the titration experiment,practical data-treatment methods and estimation ofstatistical errors. This document is described indetail using the basic level of mathematics,statistics, and programs of spreadsheet software.Especially, the titration experiments by means ofUV-visible and NMR spectroscopy are carried out anddescribed.     

DNA‐Binding Properties of Amidine‐Substituted Spiropyran Photoswitches

Two amidine‐substituted spiropyran derivatives have been characterized with respect to the DNA‐binding properties over a broad pH interval. The two derivatives differ in the number of positive charges. By varying the pH, the protonation state of the derivatives is also changed, allowing for additional variations in the charge distribution. We show that the closed spiro isomer does not bind for either of the two derivatives, whereas the open merocyanine forms bind both in the protonated and in the nonprotonated state, but with dramatically different binding constants. Flow‐oriented linear dichroism (LD) measurements also show that there are differences in the binding modes between the various forms. We rationalize these differences in terms of structure and charge distribution.     

Using the Intrinsic Fluorescence of DNA to Characterize Aptamer Binding

The reliable, readily accessible and label-free measurement of aptamer binding remains a challenge in the field. Recent reports have shown large changes in the intrinsic fluorescence of DNA upon the formation of G-quadruplex and i-motif structures. In this work, we examined whether DNA intrinsic fluorescence can be used for studying aptamer binding. First, DNA hybridization resulted in a drop in the fluorescence, which was observed for A30/T30 and a 24-mer random DNA sequence. Next, a series of DNA aptamers were studied. Cortisol and Hg²⁺ induced fluorescence increases for their respective aptamers. For the cortisol aptamer, the length of the terminal stem needs to be short to produce a fluorescence change. However, caffeine and adenosine failed to produce a fluorescence change, regardless of the stem length. Overall, using the intrinsic fluorescence of DNA may be a reliable and accessible method to study a limited number of aptamers that can produce fluorescence changes.     

Computational Characterization of Binding of Small Molecule Inhibitors to HIV‐1 gp41

Developing orally available small molecule inhibitors of HIV‐1 fusion has attracted significant interest over many years. Frey had recently reported several synthetic compounds which are experimentally shown to inhibit cell‐cell fusion in the low micromolar range. We carried out computational study to help identify possible binding modes by docking these compounds onto the hydrophobic pocket on gp41 and to characterize structures of binding complexes. The detailed gp41‐molecule binding interactions and free energies of binding are obtained through molecular dynamics simulation and MM‐PBSA calculation. Specific molecular interactions in the gp41‐inhibitor complexes are identified. The present computational study complements the corresponding experimental investigation and helps establish a good starting point for further refinement of small molecular gp41 inhibitors.     

内吗啡肽-1及其类似物的合成及与阿片受体结合作用

内吗啡肽－1和内吗啡肽－2（Endomorphin-1,EM-1;Endomorphin-2,EM-2)是阿片肽类化合物中近期发现的两种,被认为是μ型阿片受体（μ-opiate receptor,MOR)的高亲和性、高选择性的内源性配基^[1]。这一发现是阿片肽研究领域的又一次重大突破,尤其是EM－1与阿片受体相结合的作用^[2]、镇痛效应和降血压活性引起了广泛关注。Fiori等^[3]进而指出了EM－1的生物活性构象,当其处于膜模拟环境中时,认为主链采取伸展构象,2－Pro等^[3]进而指出了EM－1的生物活性构象,当其处于膜模拟环境中时,认为主链采取伸展构,2－Pro采取顺式构型;1－Tyr的侧链与3－Trp的侧链距离较近,而结合EM－1构象研究成果,合理替换了EM－1的2－／3－位氨基酸（Amino acid,Aa),用液相多肽合成法得到了EM－1及其6个类似物,并利用离体生物检定法研究了它们的阿片受体结合作用,以期有助于内吗啡肽进一步的基础及应用研究。     

金属离子与氨羧类螯合配体的键合特性研究

利用前沿色谱法,通过Cu2+、Ni2+和Co2+与螯合配体IDA 键合的准确度(R2>0.98)与精密度(RSD <5%)实验,研究了前沿色谱法同时测定络合稳定常数KML与总键合位点数Λ0值的可行性.为了进一步证明前沿色谱法的普适性,以Cu2+、Ni2+和Co2+为代表,在3种键合缓冲体系(NaAc-HAc、Na-PB、Tris-HCl)中,考察了金属离子在3种氨羧类螯合配体(IDA、Asp、Glu)上络合稳定常数KML的变化规律.结果表明,螯合配体对金属离子键合强度遵循IDA>Asp>Glu;金属离子对螯合配体键合强度遵循Cu2+>Ni2+>Co2+;3种键合缓冲体系中,NaAc-HAc键合效果最好.按照M06/6-311++G (d, p) 方法对螯合配体与金属离子的结合能(ΔE)与吉布斯自由能(ΔG)进行相关的量子计算.根据ΔE 与ΔG 的大小,从理论上推测出螯合配体与金属离子的键合规律,此规律与上述实验结果基本相符.本研究为金属离子与螯合配体间键合参数的求取提供了有效的方法和手段,从而为今后提高IMAC柱的稳定性,解决固定金属亲和柱在应用过程中尤其是竞争洗脱过程中金属离子的流失问题奠定了良好的基础.