化学交联在蛋白质结构研究中的应用

在交联剂分子两端各有一个相同或不同的活性基团,它们能与蛋白质侧链上的氨基、巯基、羟基等形成共价交联.利用交联反应,可以测定寡聚蛋白质的亚基数量、研究蛋白质的高级结构、测量氨基酸残基间的距离及研究蛋白质间的相互作用.     

酪氨酸酶、漆酶和谷氨酰胺转氨酶催化交联蛋白质的比较分析

蛋白质交联在食品加工、组织工程、酶工程和药物传递等领域具有广泛用途。以酪蛋白和牛血清白蛋白(BSA)为模式蛋白,考察酪氨酸酶、漆酶和谷氨酰胺转氨酶催化蛋白质交联的底物特异性及交联规律,揭示酶对底物蛋白质结构及反应条件的要求。采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析酶催化蛋白质交联规律,激光粒度分布仪测量交联产物粒径。结果表明：酪氨酸酶、漆酶和谷氨酰胺转氨酶对底物的特异性有共同特征,即均可以催化结构松散的蛋白质分子(酪蛋白)交联,但不能催化结构紧密的蛋白质分子(BSA)交联;还原剂二硫苏糖醇(DTT)的加入能促进酶催化BSA交联反应;DTT对酪氨酸酶和谷氨酰胺转氨酶催化酪蛋白交联无影响,但抑制漆酶对酪蛋白的交联。     

蛋白质结构与相互作用研究新方法——交联质谱技术

蛋白质的空间结构信息以及蛋白质间的相互作用信息对于研究蛋白质的功能有重要意义.研究蛋白质结构与相互作用的传统技术,如核磁共振技术、X射线晶体衍射技术等,对于蛋白质的纯度、结晶性和绝对量均有比较高的要求,限制了其广泛应用.交联质谱技术是近十多年来发展起来的新技术,它将质谱技术与交联技术相结合,在研究蛋白质结构与相互作用方面具有速度快、成本小、蛋白质各方面性状要求低等优势.本文就交联质谱技术各个环节的技术方法加以综述,包括交联质谱实验分离富集技术、常见交联剂特性、交联质谱数据库搜索算法、结果验证研究和交联质谱技术的应用等方面,并展望了该研究方向未来的发展.     

交联法在分子生物学研究中的应用

本文概要介绍了交联法用于阐明染色质、核小体、病毒粒子、核糖体结构、观察HS70基因-组蛋白等七种核酸-蛋白质复合物中大分子相互作用的实例;列举了它在DNA构象、RNA立体结构及mRNA、tRNA、snRNA、hnRNA、rRNA间相互作用等研宄中的应用。从文中可以看出,交联法的应用范围遍及遗传信息储存的高级结构、转录体系及调控、翻译系统及调控等分子生物学研究的几个主要侧面。     

化学交联质谱技术的研究进展

化学交联质谱技术是解析蛋白质结构和研究蛋白质相互作用的重要工具。近5年以来,该技术在方法和应用上都取得了很大的进步。方法上,一方面可断裂交联剂与新型分离富集方法展现了较好的应用前景,另一方面更加高效的交联肽段搜索引擎和质量控制方法为交联质谱数据分析提供了有力的工具。应用上,一方面与冷冻电镜技术结合解析了大量蛋白质的结构,另一方面从研究蛋白质复合物的相互作用发展到研究全蛋白质组水平的相互作用网络。化学交联质谱技术在方法和应用上的蓬勃发展,体现了这一技术的重要作用。本文对化学交联质谱技术的各个环节进行了详细的综述,包括交联剂选择、交联反应、酶切、交联肽段富集、液质联用、交联肽段鉴定、质量控制和生物学应用,重点介绍了最近5年的研究进展。最后,讨论了化学交联质谱技术面临的挑战及未来的发展方向。     

体内甲醛交联及染色质免疫沉淀--研究DNA和蛋白质作用的新方法

甲醛交联及染色质免疫沉淀作用研究体内DNA和蛋白质相互作用的一种新方法,在染色质结构研究中获得了广泛的应用。该方法利用甲醛固定活细胞中的DNA与蛋白质,通过免疫沉淀分离复合物,从而分析蛋白质及其体内的DNA结合序列。     

自噬和泛素-蛋白酶体系统之间的交联

自噬和泛素-蛋白酶体系统作为细胞内最重要的两大降解途径,对细胞稳态及细胞正常生理功能的维持都具有十分重要的作用。目前,越来越多的证据显示,这两大降解途径之间存在多种交联方式。首先,自噬和泛素-蛋白酶体系统都能以泛素作为共同标签,从而将泛素化底物降解;其次,泛素化的蛋白酶体可以通过自噬被清除,自噬相关蛋白质也可以通过蛋白酶体系统被降解;再次,这两条途径在细胞内能协同降解同一种底物;最后,它们之间可以相互调节活性,任一条途径被干扰都将影响另一条途径的活性。自噬和泛素-蛋白酶体系统之间的交联对细胞稳态的维持至关重要。交联失调不仅导致细胞功能异常,还可引起多种疾病的发生。本文主要对自噬和泛素-蛋白酶体系统之间的交联方式及其分子机制进行阐述,有助于深入了解细胞的分解代谢过程,进一步理解细胞稳态的维持机制,继而加深对相关疾病病理机制的认识。     

用碱洗脱法检测DNA-蛋白质交联和DNA链间交联

本方法以DNA单链断裂的检测为基础,在背景γ射线照射下进行DNA交联检测。所建方法与Kohn氏原法相比,洗脱时间大为缩短,实验所用主要材料都能立足国内。本文引入“交联度”这个参数,能同时相对定量地表示DNA总交联、DNA-蛋白质交联和DNA链间交联。此外还从DNA、蛋白质两方面确证了DNA-蛋白质交联的存在。     

功能蛋白微阵列的作用

对蛋白质阵列的两大功能—分子识别和酶活测定进行了阐述.其中前者包括蛋白质-D N A间的相互作用、蛋白质-蛋白质复合体的相互作用、蛋白质-小分子之间的相互作用;后者包括阵列蛋白作为底物来测酶活、阵列蛋白作为酶的酶活.     

菌紫质膜对315nm辐照的光反应

利用仪器本身的测量光束３１５ｎｍ光照对紫膜薄膜中菌紫质的光反应的影响的ＣＤ谱研究说明：３１５ｎｍ的近紫外光可以激发薄膜中菌紫质的光反应,３１５ｎｍ与４０８ｎｍ、３３５ｎｍ光激发的光反应变化类型一致,但与５６８ｎｍ光激发的反应变化类型不一致;３１５ｎｍ光激发的光反应与菌紫质的初始样品状态有关,与菌紫质所处的分子状态的分布有关,而不是直接与初始样品状态存在的表现条件有关。结果认为利用包括近ＵＶ光在内的不同光照条件来调控ＢＲ的光反应是有可能的。     

Electron‐deficient p‐benzoyl‐l‐phenylalanine derivatives increase covalent chemical capture yields for protein–protein interactions

The photoactivatable amino acid p‐benzoyl‐l ‐phenylalanine (pBpa) has been used for the covalent capture of protein–protein interactions (PPIs) in vitro and in living cells. However, this technique often suffers from poor photocrosslinking yields due to the low reactivity of the active species. Here we demonstrate that the incorporation of halogenated pBpa analogs into proteins leads to increased crosslinking yields for protein–protein interactions. The analogs can be incorporated into live yeast and upon irradiation capture endogenous PPIs. Halogenated pBpas will extend the scope of PPIs that can be captured and expand the toolbox for mapping PPIs in their native environment.     

Protein S3 in the human 80S ribosome adjoins mRNA 3′ of the A-site codon

The protein environment of mRNA 3′ of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5′-end and a perfluoroarylazide group at one of the nucleotide residues 3′ of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNA^Phe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNA^Phe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in ternary complexes and in the absence of tRNA. Within ternary complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed; it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3′ of the codon in the decoding site.     

Agonism and Antagonism at the Insulin Receptor

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (³H-thymidine incorporation), and not on metabolic endpoints (¹⁴C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (³H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.     

Internucleotide movements during formation of 16 S rRNA-rRNA photocrosslinks and their connection to the 30 S subunit conformational dynamics

UV light-induced RNA photocrosslinks are formed at a limited number of specific sites in the Escherichia coli and in other eubacterial 16 S rRNAs. To determine if unusually favorable internucleotide geometries could explain the restricted crosslinking patterns, parameters describing the internucleotide geometries were calculated from the Thermus thermophilus 30 S subunit X-ray structure and compared to crosslinking frequencies. Significant structural adjustments between the nucleotide pairs usually are needed for crosslinking. Correlations between the crosslinking frequencies and the geometrical parameters indicate that nucleotide pairs closer to the orientation needed for photoreaction have higher crosslinking frequencies. These data are consistent with transient conformational changes during crosslink formation in which the arrangements needed for photochemical reaction are attained during the electronic excitation times. The average structural rearrangement for UVA-4-thiouridine (s4U)-induced crosslinking is larger than that for UVB or UVC-induced crosslinking; this is associated with the longer excitation time for s4U and is also consistent with transient conformational changes. The geometrical parameters do not completely predict the crosslinking frequencies, implicating other aspects of the tertiary structure or conformational flexibility in determining the frequencies and the locations of the crosslinking sites. The majority of the UVB/C and UVA-s4U-induced crosslinks are located in four regions in the 30 S subunit, within or at the ends of RNA helix 34, in the tRNA P-site, in the distal end of helix 28 and in the helix 19/helix 27 region. These regions are implicated in different aspects of tRNA accommodation, translocation and in the termination reaction. These results show that photocrosslinking is an indicator for sites where there is internucleotide conformational flexibility and these sites are largely restricted to parts of the 30 S subunit associated with ribosome function.     

Protein S3 fragments neighboring mRNA during elongation and termination of translation on the human ribosome

Protein S3 fragments were determined that crosslink to modified mRNA analogues in positions +5 to +12 relative to the first nucleotide in the P-site bound codon in model complexes mimicking states of ribosomes at the elongation and translation termination steps. The mRNA analogues contained a Phe codon UUU/UUC at the 5′-termini that could predetermine the position of the tRNA^Phe on the ribosome by the P-site binding and perfluorophenylazidobenzoyl group at a nucleotide in various positions 3′ of the UUU/UUC codon. The crosslinked S3 protein was isolated from 80S ribosomal complexes irradiated with mild UV light and subjected to cyanogen bromide—induced cleavage at methionine residues with subsequent identification of the crosslinked oligopeptides. An analysis of the positions of modified oligopeptides resulting from the cleavage showed that, in dependence on the positions of modified nucleotides in the mRNA analogue, the crosslinking sites were found in the N-terminal half of the protein (fragment 2–217) and/or in the C-terminal fragment 190–236; the latter reflects a new peculiarity in the structure of the mRNA binding center in the ribosome, unknown to date. The results of crosslinking did not depend on the type of A-site codon or on the presence of translation termination factor eRF1.     

Synthesis of six novel N,N-dialkyl derivatives of spermidine and effects on growth of the fungal plant pathogen Pyrenophora avenae

Six novel N,N-dialkyl derivatives of spermidine were synthesised and examined for activity against the oat stripe pathogen Pyrenophora avenae. Two of these spermidine analogues, N,N-dimethyl-N1-(3-aminopropyl)-1,3-diaminopropane trihydrochloride (27) and N,N-dimethyl-N1-(3-aminopropyl)-1,4-diaminobutane trihydrochloride (28), reduced radial extension of P. avenae on plates when used at 2 mM, and caused more substantial reductions in fungal growth in liquid culture when used at 1 mM. Preliminary data suggest that neither compound affected polyamine biosynthesis, determined by following the incorporation of label from ornithine into polyamines and examining intracellular polyamine concentrations in fungal tissue.     

Characterization of the cellular uptake and metabolic conversion of acetylated N-acetylmannosamine (ManNAc) analogues to sialic acids

"Sialic acid engineering" refers to the strategy where cell surface carbohydrates are modified by the biosynthetic incorporation of metabolic intermediates, such as non-natural N-acetylmannosamine (ManNAc) analogues, into cellular glycoconjugates. While this technology has promising research, biomedical, and biotechnological applications due to its ability to endow the cell surface with novel physical and chemical properties, its adoption on a large scale is hindered by the inefficient metabolic utilization of ManNAc analogues. We address this limitation by proposing the use of acetylated ManNAc analogues for sialic acid engineering applications. In this paper, the metabolic flux of these "second-generation" compounds into a cell, and, subsequently, into the target sialic acid biosynthetic pathway is characterized in detail. We show that acetylated ManNAc analogues are metabolized up to 900-fold more efficiently than their natural counterparts. The acetylated compounds, however, decrease cell viability under certain culture conditions. To determine if these toxic side effects can be avoided, we developed an assay to measure the cellular uptake of acetylated ManNAc from the culture medium and its subsequent flux into sialic acid biosynthetic pathway. This assay shows that the majority ( > 80%) of acetylated ManNAc is stored in a cellular "reservoir" capable of safely sequestering this analogue. These results provide conditions that, from a practical perspective, enable the acetylated analogues to be used safely and efficaciously and therefore offer a general strategy to facilitate metabolic substrate-based carbohydrate engineering efforts. In addition, these results provide fundamental new insights into the metabolic processing of non-natural monosaccharides.     

Lipophilic prodrug conjugates allow facile and rapid synthesis of high-loading capacity liposomes without the need for post-assembly purification

Abstract

Dihydropyridopyrazoles are simplified synthetic analogues of podophyllotoxin that can effectively mimic its molecular scaffold and act as potent mitotic spindle poisons in dividing cancer cells. However, despite nanomolar potencies and ease of synthetic preparation, further clinical development of these promising anticancer agents is hampered due to their poor aqueous solubility. In this article, we developed a prodrug strategy that enables incorporation of dihydropyridopyrazoles into liposome bilayers to overcome the solubility issues. The active drug was covalently connected to either myristic or palmitic acid anchor via carboxylesterase hydrolyzable linkage. The resulting prodrugs were self-assembled into liposome bilayers from hydrated lipid films using ultrasound without the need for post-assembly purification. The average particle size of the prodrug-loaded liposomes was about 90?nm. The prodrug incorporation was verified by differential scanning calorimetry, spectrophotometry and gel filtration reaching maximum at 0.3 and 0.35 prodrug/lipid molar ratios for myristic and palmitic conjugates, respectively. However, the ratio of 0.2 was used in the particle size and biological activity experiments to maintain long-term stability of the prodrug-loaded liposomes against phase separation during storage. Antiproliferative activity was tested against HeLa and Jurkat cancer cell lines in vitro showing that the liposomal prodrug retained antitubulin activity of the parent drug and induced apoptosis-mediated cancer cell death. Overall, the established data provide a powerful platform for further clinical development of dihydropyridopyrazoles using liposomes as the drug delivery system.     

Docking Studies of Adenosine Analogues with NS5 Methyltransferase of Yellow Fever Virus

Yellow fever virus (YFV) is caused by single stranded positive RNA virus called Flavivirus. Till now no specific antiviral agents are available for the treatment of YFV, and despite a commercial YFV vaccine, there are still approximately 30,000 deaths worldwide each year and cases have been increasing in the last 20 years. Here, the effects of adenosine analogues and commercially available adenosine derivative drugs on NS5 methyltransferase of YFV have been performed by the comparative docking study. Based on the docking score, the glide energy and the number of interactions of the adenosine analogues with the Pubchem ID 13792 and 1077 showed the better scoring function than the best ranked commercially available adenosine analogue derived antiviral drug Cc3ado. From the docking result it reveals that these adenosine analogues can bind to the active site of NS5 methyltransferase protein and inhibit the viral replication.     

Template Location on the Human Ribosome: Environment of the mRNA Nucleotide Adjacent to the A-Site Codon on the 3′-Side

The 18S rRNA nucleotides close to the template nucleotide adjacent to the 80S ribosomal A-site codon on the 3′-end (i.e., the nucleotide in position +7 relative to the first nucleotide of the P-site codon) were identified using the affinity crosslinking approach. For this purpose, the photoreactive mRNA analogues with a perfluorophenylazide group attached through various linkers to the uridine C5, 3′-terminal phosphate or guanosine N7 were used. The position of the mRNA analogues on the ribosome was preset using tRNA^Phe, which recognized the phenylalanine codon directed to the P-site. An analysis of the rRNAs isolated from the irradiated complexes of 80S ribosomes showed that all the analogues are almost equally crosslinked to the 18S rRNA nucleotides we attributed to the A-site codon environment: namely, to nucleotides A1823, A1824, and A1825 of the 3′-minidomain and to the 620–630 fragment of the 18S rRNA 5′-domain. In addition, we identified a new component of the mRNA binding site of human ribosomes, nucleotide C1698, belonging to the 18S rRNA 3′-minidomain, using analogues bearing a perfluorophenylazide group on uridine and guanine residues.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 295–302.Original Russian Text Copyright © 2005 by Demeshkina, Styazhkina, Bulygin, Repkova, Ven’yaminova, Karpova.