泛素化蛋白的化学合成及半合成

蛋白质翻译后修饰（post-translational modifications,PTMs）在调节蛋白质的结构和功能上发挥着重要作用,异常的蛋白质翻译后修饰会导致某些疾病的发生.泛素化（ubiquitination）作为一类重要的蛋白质翻译后修饰,已经被证明与细胞的稳态以及细胞内部的多条信号通路有关,主要参与细胞内蛋白的定位、调节和降解以及细胞周期、基因表达、信号传递、损伤修复、炎症免疫等多种生命过程.然而,对于泛素蛋白的相关研究虽然进行了多年,但是许多重要的科学问题尚未研究清楚,其中如何高效地获得大量、均一的泛素化蛋白是最重要的挑战之一.近年来,蛋白质化学合成及半合成手段的发展,很大程度上解决了这一难题.本文主要针对目前常用的泛素化蛋白的化学合成和半合成方法进行综述.     

基于靶向多肽探针的蛋白质分析检测新方法

高选择性、高灵敏度的蛋白质分析检测方法是复杂生命体系研究的有力工具,能为疾病的发现、治疗以及分子机制研究提供新的、有价值的思路和手段.该文围绕蛋白质与多肽的分子识别,针对疾病相关多肽、蛋白质的分离、分析与检测新方法,综述了近几年取得的研究进展,可为复杂生命体系中生理活性分子的结构与功能研究提供借鉴.     

亲和毛细管电泳技术在蛋白质-DNA相互作用研究中的应用

蛋白质-DNA的相互作用在决定细胞命运的许多过程中发挥重要作用,对蛋白质-DNA相互作用的分子机制研究有利于对基本生命过程的理解,为相关疾病的临床治疗及药物筛选提供理论指导。另一方面,利用一些已知的蛋白质-DNA相互作用可以帮助开发先进的生物工程和生命分析技术,为相关研究提供有力的技术支持。因此,建立灵敏、快速的分析方法用于表征蛋白质-DNA的相互作用十分重要。高效毛细管电泳（capillary electrophoresis,CE）技术因其超高的分离效率、极低的样品消耗与较短的分析时间等优势被广泛应用于化学、生命科学和环境科学等多个研究领域。其中,亲和毛细管电泳（affinity capillary electrophoresis,ACE）技术已经成为考察分子间相互作用的重要研究工具。这篇文章综述了亲和毛细管电泳技术自建立以来在蛋白质-DNA相互作用分析方面的研究进展,并对经典的研究工作进行了着重介绍,主要包括三方面的内容：（1）亲和毛细管电泳技术简介;（2）利用亲和毛细管电泳技术进行蛋白质-DNA相互作用的基础分子机制研究;（3）利用已知的蛋白质-DNA相互作用发展针对目标分子及目标反应的亲和毛细管电泳检测技术。本文还对该领域的未来发展趋势进行了展望与探讨,提出应从以下两个方面增强亲和毛细管电泳技术的分析能力：（1）充分发挥CE技术样品消耗少和高通量等优势,分别发展针对少量珍贵生物样品的高灵敏检测方法和针对大量未知因素的高通量筛选方法;（2）结合DNA测序及质谱技术快速筛选、鉴定未知的蛋白质-DNA相互作用的精确靶点。     

泛素化核小体复合物的结构机制研究进展

核小体组蛋白(H2A、H2B、H3、H4)的泛素化修饰介导了一系列效应蛋白与核小体发生特异性的相互作用,并调控细胞的表观遗传.近年来得益于一些新的化学方法与生物方法的发展,如蛋白质化学修饰等样品获取技术以及冷冻电子显微镜等结构观测技术,一些泛素化核小体与效应蛋白的复合物结构得以被解析,揭示了泛素调控染色质表观遗传过程的分子机制.本文对近期报道的六种泛素化核小体与效应蛋白的复合物进行了归纳,对比分析了泛素化核小体对效应蛋白的特异性识别与活化机制,并总结了组蛋白上的关键相互作用位点.     

氢氘交换质谱技术在蛋白质和蛋白复合物结构研究中的应用进展

蛋白质是生命功能的执行者,其功能的发挥受自身结构动态变化、与其他生物分子的相互作用及修饰等因素的调节。因此,对蛋白质及蛋白复合物结构的研究有助于揭示重要生命过程中的分子机理与机制。氢氘交换质谱(Hydrogen deuterium exchange mass spectrometry,HDX-MS)是研究蛋白质结构、动态变化和相互作用的强有力工具,也是传统生物物理手段的重要补充。该文综述了HDX-MS的基本原理、机制、实验方法和研究最新进展,并从蛋白质自身动态变化、蛋白质-小分子相互作用、蛋白质-蛋白质相互作用3个方面介绍了近年来HDX-MS在蛋白及蛋白复合物研究中的应用进展。     

核酸适体靶向的膜蛋白识别与功能调控研究进展

膜蛋白在细胞生命活动中发挥着重要作用, 研究并调控细胞膜蛋白的结构和功能有助于阐明生命活动的基本规律, 为新型药物研发和高效疾病诊治提供研究基础. 核酸适体是一类特殊的寡核苷酸序列, 因具有特异性识别靶标的能力而被广泛用于生物传感领域. 将核酸适体与DNA纳米技术相结合, 利用DNA分子可程序化设计、 可功能化修饰等优势, 发展核酸适体靶向的膜蛋白识别与功能调控方法可为研究膜蛋白相互作用提供有力工具. 本文介绍了基于核酸适体靶向识别的DNA纳米技术在膜蛋白识别及细胞功能调控中的研究进展, 并对核酸适体靶向的膜蛋白识别及功能调控领域面临的挑战进行了分析, 对其应用前景进行了展望.     

DCN1-UBE2M相互作用抑制剂的3D-QSAR、分子对接和分子动力学研究

类泛素化是一种蛋白质翻译后修饰,其异常会导致神经退行性疾病和多种肿瘤的发生,因此它被视为有希望的抗肿瘤靶标。研究表明,抑制DCN1-UBE2M相互作用可选择性阻遏类泛素化。本文基于哌啶基脲类DCN1-UBE2M相互作用抑制剂进行3D-QSAR、分子对接和分子动力学模拟研究。利用3D-QSAR中的CoMFA和CoMSIA方法构建了相关模型,其交叉验证系数q~2分别为0.686、0.682,拟合验证系数r~2分别为0.966、0.931,表明模型是可靠的且预测能力较好。接着运用分子对接分析哌啶基脲类化合物与DCN1的相互作用,结果表明,它们主要通过氢键和疏水作用与靶蛋白结合。通过分子动力学模拟研究进一步了解结合模型和验证对接结果。本文所得的研究结果可为今后此类化合物的结构优化提供有效信息。     

智能聚合物基材料富集磷酸化肽和糖肽的研究进展

翻译后修饰是蛋白质组学研究的前沿和重点,它不仅调节着蛋白质的折叠、状态、活性、定位以及蛋白质间的相互作用,也能帮助科学家更全面地了解生物体的生命过程,为疾病的预测、诊断和治疗提供更加强大的支撑和依据。翻译后修饰产物(例如磷酸化肽和糖肽)丰度很低,且存在着强烈的背景干扰,很难直接用质谱进行分析,因此迫切需要开发高效的富集材料和技术来选择性富集翻译后修饰产物。近年来,智能聚合物基材料通过外部物理、化学或生物刺激可逆地改变其结构和功能,实现对磷酸化肽和糖肽高度可控的吸附和脱附,进而衍生开发出一系列新颖的富集方法,极大地吸引研究者们的兴趣。一方面,智能聚合物基材料的响应变化包括材料疏水性的增加或减少、形状和形貌的改变、表面电荷的重新分布以及亲和配体的暴露或隐藏等特性。这些特性使得目标物和智能聚合物基材料之间的亲和力可以通过简单改变外部条件(如温度、pH值、溶剂极性和生物分子等)实现更可控和更智能的精细调节。另一方面,智能聚合物基材料为集成功能模块提供了便捷的可扩展平台,例如特定的识别组件,显著提高了目标物质的分离选择性。智能聚合物基材料在分离方面展现出巨大的潜力,这为蛋白质翻译后修饰产物的分析和研究带来了希望。围绕上述主题,该文依据Web of Science近20年来近50篇代表性文献,概述了智能聚合物基材料在磷酸化肽和糖肽分离及富集中的发展方向。     

蛋白质甲基化修饰的蛋白质组学分析方法新进展

蛋白质的甲基化修饰是一类重要的翻译后修饰。但与磷酸化、糖基化和泛素化等翻译后修饰相比,甲基化修饰的蛋白质组学分析方法开发还是一个较新的研究领域。近几年,由于甲基化修饰在表观遗传调控中的重要作用,这一修饰类型得到了越来越多的关注,相关的分析技术和分析方法也取得了较多进展。其中,基于质谱的蛋白质组学分析方法在甲基化修饰中发挥着关键的作用,实现了这一甲基化修饰的高通量分析。该综述将从甲基化修饰的分离富集、假阳性率控制以及定量蛋白质组学等方面对一些蛋白质甲基化修饰的分析技术和方法的最新进展进行介绍。     

蛋白质翻译后脂修饰的结构和功能分析:靶向细胞脂信号

蛋白质翻译后脂修饰是指蛋白质在核糖体合成后与疏水脂质分子的共价结合.在已知共价化学修饰中,脂质分子独特的物理化学性质赋予了蛋白质特殊的结构和功能,并极大地影响蛋白质的膜锚定能力、转运和定位途径、信号转导以及蛋白质相互作用等.多样化的脂质结构和结合方式决定了脂修饰蛋白功能的复杂性,这些脂修饰蛋白与其他已知或未知修饰蛋白一起,在细胞多层次交叉调控信号转导通路中互相影响,协同作用,从而实现对生理活动的精细调控.开展蛋白质翻译后脂修饰结构与功能分析是揭示微生物感染、免疫调节、肿瘤发生发展等机制的重要途径,对发现和筛选疾病标志物以及挖掘新型药物靶标具有重要意义.     

Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

Preparing to read the ubiquitin code: a middle‐out strategy for characterization of all lysine‐linked diubiquitins

Multiple studies demonstrate that ubiquitination of proteins codes for regulation of cell differentiation, apoptosis, endocytosis and many other cellular functions. There is great interest in and considerable effort being given to defining the relationships between the structures of polyubiquitin modifications and the fates of the modified proteins. Does each ubiquitin modification achieve a specific effect, much like phosphorylation, or is ubiquitin like glycosylation, where there is heterogeneity and redundancy in the signal? The sensitive analytical tools needed to address such questions readily are not yet mature. To lay the foundation for mass spectrometry (MS)‐based studies of the ubiquitin code, we have assembled seven isomeric diubiquitins with all‐native sequences and isopeptide linkages. Using these compounds as standards enables the development and testing of a new MS‐based strategy tailored specifically to characterize the number and sites of isopeptide linkages in polyubiquitin chains. Here, we report the use of Asp‐selective acid cleavage, separation by reverse phase high‐performance liquid chromatography and characterization by tandem MS to distinguish and characterize all seven isomeric lysine‐linked ubiquitin dimers. Copyright © 2014 John Wiley & Sons, Ltd.     

Protein ubiquitination and formation of polyubiquitin chains without ATP,E1 and E2 enzymes

Studying protein ubiquitination is difficult due to the complexity of the E1–E2–E3 ubiquitination cascade. Here we report the discovery that C-terminal ubiquitin thioesters can undergo direct transthiolation with the catalytic cysteine of the model HECT E3 ubiquitin ligase Rsp5 to form a catalytically active Rsp5∼ubiquitin thioester (Rsp5∼Ub). The resulting Rsp5∼Ub undergoes efficient autoubiquitination, ubiquitinates protein substrates, and synthesizes polyubiquitin chains with native Ub isopeptide linkage specificity. Since the developed chemical system bypasses the need for ATP, E1 and E2 enzymes while maintaining the native HECT E3 mechanism, we named it “Bypassing System” (ByS). Importantly, ByS provides direct evidence that E2 enzymes are dispensable for K63 specific isopeptide bond formation between ubiquitin molecules by Rsp5 in vitro. Additionally, six other E3 enzymes including Nedd4-1, Nedd4-2, Itch, and Wwp1 HECT ligases, along with Parkin and HHARI RBR ligases processed Ub thioesters under ByS reaction conditions. These findings provide general mechanistic insights on protein ubiquitination, and offer new strategies for assay development to discover pharmacological modulators of E3 enzymes.     

Structural aspects of ubiquitin binding specificities

Ubiquitin (Ub) is widely distributed in eukaryotic cells as its name means. There are many kinds of Ub-like proteins (for example, SUMO, NEDD8 and ISG15) and Ub-like domains (UbLs) included in multi-domain proteins. To date, a large number of Ub-binding domains (UBDs), such as UBA, CUE, UIM, ZnF, and Pru, are coming up to us with different affinities to Ub and its homologues. The binding specificities provide the basis for controlling various cellular events as well as for delivering ubiquitinated proteins to proteasome for degradation. Structural details of these UBDs and their complexes with Ub might as well show us the delicate mechanism of Ub recognition and regulation. This review summarizes recent progresses on deciphering the structure-based Ub-binding specificities, which are the importantly fundamental elements in orchestrating the ubiquitination and deubiquitination processes in eukaryotic cells.     

Multifunctional fractionation of polyclonal antibodies by immunoaffinity high-performance monolithic disk chromatography

High-performance monolithic disk chromatography (HPMDC), including its affinity mode, is a very efficient method for fast separations of biological molecules of different sizes and shapes. In this paper, protein and peptide ligands, immobilized on the inner surface of thin, monolithic supports (Convective Interaction Media or CIM disks), have been used to develop methods for fast, quantitative affinity fractionation of pools of polyclonal antibodies from blood sera of rabbits, immunized with complex protein-peptide conjugates. The combination of several disks with different affinity functionalities in the same cartridge enables the separation of different antibodies to be achieved within a few minutes. The apparent dissociation constants of affinity complexes were determined by frontal analysis. Variation of elution flow rate over a broad range does not affect the affinity separation characteristics. Indifferent synthetic peptides used as biocompatible spacers do not change the affinity properties of the ligands. The highly reproducible results of immunoaffinity HPMDC are compared with data obtained by widely used enzyme-linked immunosorbent assay.     

Preparing to read the ubiquitin code: characterization of ubiquitin trimers by top‐down mass spectrometry

The profound effects of ubiquitination on the movement and processing of cellular proteins depend exquisitely on the structures of monoubiquitin and polyubiquitin modifications. Unconjugated polyubiquitins also have a variety of intracellular functions. Structures and functions are not well correlated yet, because the structures of polyubiquitins and polyubiquitin modifications of proteins are difficult to decipher. We are moving towards a robust strategy to provide that structural information. In this report electron transfer dissociation mass spectra of six synthetic ubiquitin trimers (multiply branched proteins with molecular masses exceeding 25 600 Da) are examined using an Orbitrap Fusion Lumos instrument to determine how top‐down mass spectrometry can characterize the chain topology and linkage sites in a single, facile workflow. The efficacy of this method relies on the formation, detection, and interpretation of extensive fragmentation. Copyright © 2016 John Wiley & Sons, Ltd.     

Probing affinity and ubiquitin linkage selectivity of ubiquitin-binding domains using mass spectrometry

Non-covalent interactions between ubiquitin (Ub)-modified substrates and Ub-binding domains (UBDs) are fundamental to signal transduction by Ub receptor proteins. Poly-Ub chains, linked through isopeptide bonds between internal Lys residues and the C-terminus of Ub, can be assembled with varied topologies to mediate different cellular processes. We have developed and applied a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to determine isopeptide linkage-selectivity and affinity of poly-Ub·UBD interactions. We demonstrate the technique using mono-Ub and poly-Ub complexes with a number of α-helical and zinc-finger (ZnF) UBDs from proteins with roles in neurodegenerative diseases and cancer. Affinities in the 2-200 μM range were determined to be in excellent agreement with data derived from other biophysical techniques, where available. Application of the methodology provided further insights into the poly-Ub linkage specificity of the hHR23A-UBA2 domain, confirming its role in Lys48-linked poly-Ub signaling. The ZnF UBP domain of isopeptidase-T showed no linkage specificity for poly-Ub chains, and the Rabex-5 MIU also exhibited little or no specificity. The discovery that a number of domains are able to bind cyclic Lys48 di-Ub with affinities similar to those for the acyclic form indicates that cyclic poly-Ub may be capable of playing a role in Ub-signaling. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multi-site interactions, opening the way for the study of crosstalk between individual Ub-signaling pathways.     

Patents and literature

The separation and purification of biologically functional molecules (e.g., proteins, antibodies, peptides, hormones, low molecular weight biologicals) is of fundamental importance to biotechnology. Affinity separations have become a particularly attractive method for bioseparations due to their high degree of selectivity. Numerous affinity ligands have been prepared in recent years including lectins, nucleic acids, inhibitors, and immunoresponse agents. Furthermore, a variety of novel supports have been synthesized to aid in the development of commercially useful affinity separation systems. Recent US patents and scientific literature on affinity separations and purifications are surveyed. Patent abstracts are summarized individually and a list of literature references are given.     

Preparing to read the ubiquitin code: top‐down analysis of unanchored ubiquitin tetramers

The characterization of polyubiquitin chains has been an analytical challenge for several decades. It has been shown that anchored and unanchored polyubiquitin chains with different isopeptide linkages and lengths exhibit a wide range of profoundly different cellular functions. However, structure function studies have been hindered by the difficulty of characterizing these complex chain structures. This report presents a broadly applicable workflow to characterize ubiquitin tetramers without the need for genetic mutations or reiterative immunoprecipitations. We use a top‐down proteomic strategy that exploits ETciD activation on an orbitrap Fusion Lumos and manual interpretation aided by graphical interpretation of mass shifts to facilitate characterization of chain topography and lysine linkage sites. Our workflow differentiates all topological features of the numerous isomers of tetraubiquitin, which have molecular masses in excess of 34 000 Da and identifies linkage sites in these branched proteins. Copyright © 2016 John Wiley & Sons, Ltd.