去泛素化酶在乳腺癌中的研究新进展

泛素-蛋白酶体系统(ubiquitin-proteasome-system,UPS)是控制蛋白质降解的主要系统,也是细胞基本活动的关键调节器。去泛素化酶(deubiquitinating enzymes,DUBs)是泛素-蛋白酶体系统的组成部分,主要参与调节蛋白质泛素化和去泛素化的动态平衡,对细胞增殖、信号转导、神经病变或肿瘤发生意义重大。不同的DUBs在乳腺癌中的作用不同,最新发现去泛素化酶BAP1、OTUD3、ATXN3L主要调节乳腺癌细胞增殖,某些DUBs小分子抑制剂可以间接诱导三阴性乳腺癌细胞凋亡。本文主要综述这三个DUBs及去泛素化酶抑制剂在乳腺癌中的研究新进展,为寻找新型的乳腺癌分子靶向药物提供理论依据。     

泛素连接酶和去泛素化酶在帕金森病中的作用

中脑黑质多巴胺能神经元特异性损伤和α突触核蛋白聚集的分子机制是帕金森病（Parkinson’s disease,PD）研究领域亟待解决的问题。蛋白质异常聚集很大程度上是由于泛素-蛋白酶体系统（ubiquitin-proteasome system,UPS）功能障碍引起的。蛋白质泛素化由一系列泛素化酶级联反应促进,并受去泛素化酶（deubiquitylases,DUBs）的反向调节。泛素化和去泛素化过程异常导致蛋白质异常聚集和包涵体形成,进而损伤神经元。近来研究报道,蛋白质的泛素化和去泛素化修饰在PD的发病机制中发挥重要作用。E3泛素连接酶促进蛋白质的泛素化,有利于α突触核蛋白的清除、促进多巴胺能神经元的存活、维持线粒体的功能等。DUBs可以去掉底物蛋白质的泛素化修饰,抑制α突触核蛋白的降解,调控线粒体的功能和神经元内铁的稳态。本文以E3泛素连接酶和DUBs为切入点,综述了蛋白质泛素化和去泛素化修饰参与多巴胺能神经元损伤机制的最新研究进展。     

去泛素化酶在肾细胞癌中的作用

肾细胞癌（renal cell carcinoma,RCC）是成人肾脏的原发性恶性肿瘤。泛素-蛋白酶体系统（ubiquitin-proteasome system,UPS）对控制蛋白质水平和调节生理病理过程至关重要。去泛素化酶（deubiquitinases,DUBs）是UPS的关键成分,特别是从靶蛋白中去除泛素链,通过严格调节正常生理学中泛素化和去泛素化之间的平衡,对蛋白质稳态和质量控制显示出至关重要的作用。越来越多的研究表明,功能异常的DUBs与RCC的进展和转移有关。根据底物的不同,一些DUB可能会抑制RCC,而另一些则促进。本文综述了RCC相关DUB的最新研究进展,描述了其分类、功能作用,总结了DUB在RCC中的作用和作用机制,并讨论了靶向DUBs用于癌症治疗。     

泛素连接酶和去泛素化酶在阿尔茨海默病中的研究进展

阿尔茨海默病(Alzheimer's disease, AD)是一种老年人常见的中枢神经系统退行性疾病。泛素化和去泛素化过程失调导致蛋白质异常聚集是AD发生发展的主要原因。E3泛素连接酶(E3 ubiquitin ligases,E3s)调控底物蛋白的泛素化,可促进β淀粉样蛋白(amyloid-β, Aβ)和过度磷酸化Tau蛋白的清除,改善突触及神经元的功能。去泛素化酶(deubiquitinating enzymes, DUBs)去除底物蛋白的泛素化修饰,可抑制Aβ和过度磷酸化Tau蛋白的降解,引起神经炎症。因为E3s和DUBs并不通过单一途径来促进或者抑制AD的发生发展,所以本文以E3s和DUBs所属亚族为切入点,综述了E3s和DUBs在AD中作用机制的最新研究进展。     

去泛素化酶与肿瘤

泛素化修饰(ubiquitination modification)广泛存在于真核生物,通过26S蛋白酶体降解途径或信号传递等,改变蛋白质稳定性、定位和活性等功能,参与细胞的周期、转录、炎症、肿瘤和免疫等各项功能,是一类复杂的动态调控系统.泛素化调节是一个可逆过程,被泛素连接酶(ubiquitin ligase,E3)...     

去泛素化酶活性的调控

泛素化是一种非常重要的蛋白质翻译后修饰方式,在细胞生命活动的各个方面发挥作用。泛素化修饰是可逆的过程,去泛素化酶通过催化去除底物蛋白质上的泛素从而逆转该过程。去泛素化酶是一类数量众多的蛋白水解酶家族,近年来不断有新的去泛素化酶被发现和报道。鉴于其在细胞功能中的重要作用,去泛素化酶活性受到严格的调控。目前的研究表明,影响去泛素化酶活性的因素很多。本文主要从转录水平的调控、翻译后修饰、蛋白质定位和蛋白质相互作用等调控方式进行论述,以期为研究和利用去泛素化酶治疗疾病提供新思路。     

去泛素化酶USP11在细胞中的功能作用及其研究进展

Ubiquitin-specific protease 11(USP11)属于半胱氨酸蛋白酶,是去泛素化酶家族(deubiquitinating enzymes,DUBs)的重要成员之一。近年来研究表明USP11能调节细胞内众多蛋白底物的稳定性及功能,包括DNA修复蛋白、病毒RNA复制相关蛋白、TGFβ和NF-κB信号转导通路相关蛋白等,在疾病的发生发展中起着重要的作用。主要综述了USP11的结构、在细胞中的分子功能以及与肿瘤和病毒性疾病的关系,探讨了USP11作为治疗分子靶标的可能性。     

去泛素化酶在卵巢癌发生发展中的作用CSCD

泛素-蛋白酶体途径是细胞内蛋白质降解的重要方式,调节蛋白质稳定性。去泛素化酶可以识别特定蛋白质的泛素化信号从而使底物蛋白质去泛素化,逆转蛋白质泛素化过程,进而调控细胞增殖、分化、凋亡和迁移等多种生物学功能。去泛素化酶家族的多个成员通过影响细胞增殖凋亡及对化疗药物的敏感性等,在卵巢癌的发生发展中发挥十分重要的作用。一些小分子抑制剂通过抑制去泛素化酶的活性从而起到抗肿瘤的作用,并且具有特异性强,细胞毒性较弱等优势。本综述对参与卵巢癌发生发展的去泛素化酶以及相关的小分子抑制剂做一个全面的总结,为卵巢癌的诊断和治疗提供一个新的方向。     

定量蛋白质组学揭示酵母去泛素化酶Ubp14生物学功能

泛素化是一种动态可逆的蛋白质翻译后修饰,泛素分子在泛素激活酶、泛素结合酶和泛素连接酶的级联酶促反应催化下共价连接到底物蛋白上。去泛素化酶将泛素分子从底物上移除,动态可逆地调控泛素化修饰,在成熟泛素的生成、泛素链的移除与修剪、游离泛素链的回收等过程中发挥着关键的调控作用。本文的研究对象是酵母中泛素特异性蛋白酶(ubiquitin specific protease, USP)家族成员Ubp14,负责回收细胞内游离的泛素链。本研究定量比较了酵母细胞中Ubp14缺失对全蛋白质组的影响,进而找出其潜在的调控通路和分子功能。首先,通过同源重组技术构建了ubp14?菌株,发现其生长速度低于野生型酵母。利用稳定同位素氨基酸代谢标记技术结合深度覆盖的蛋白质组学分析技术,系统比较了ubp14?菌株相对于野生型菌株的差异蛋白,共计鉴定3 685个蛋白,通过统计学分析筛选得到109个差异蛋白。基因本体论分析发现,Ubp14缺失引起的差异蛋白主要参与了包括氨基酸代谢、氧化还原和热应激等生物学过程。本研究为深入探究去泛素化酶Ubp14的生物学功能,进而深刻理解游离泛素的稳态平衡与生物学过程调控提供了高可信的蛋白...     

组蛋白的泛素化与去泛素化修饰

泛素在真核生物体内广泛存在,泛素化修饰是转录后的修饰方式之一;组蛋白是染色质的主要成分之一,与基因的表达有密切关系。组蛋白的泛素化修饰与经典的蛋白质的泛素调节途径不同,不会导致蛋白质的降解,但是能够招募核小体到染色体、参与X染色体的失活、影响组蛋白的甲基化和基因的转录。组蛋白的去泛素化修饰同样与染色质的结构及基因表达密切相关。组蛋白的泛素化和磷酸化、乙酰化、甲基化修饰之间还存在协同和级联效应。     

Integration of over 9,000 mass spectrometry experiments builds a global map of human protein complexes

Macromolecular protein complexes carry out many of the essential functions of cells, and many genetic diseases arise from disrupting the functions of such complexes. Currently, there is great interest in defining the complete set of human protein complexes, but recent published maps lack comprehensive coverage. Here, through the synthesis of over 9,000 published mass spectrometry experiments, we present hu.MAP, the most comprehensive and accurate human protein complex map to date, containing > 4,600 total complexes, > 7,700 proteins, and > 56,000 unique interactions, including thousands of confident protein interactions not identified by the original publications. hu.MAP accurately recapitulates known complexes withheld from the learning procedure, which was optimized with the aid of a new quantitative metric (k‐cliques) for comparing sets of sets. The vast majority of complexes in our map are significantly enriched with literature annotations, and the map overall shows improved coverage of many disease‐associated proteins, as we describe in detail for ciliopathies. Using hu.MAP, we predicted and experimentally validated candidate ciliopathy disease genes in vivo in a model vertebrate, discovering CCDC138, WDR90, and KIAA1328 to be new cilia basal body/centriolar satellite proteins, and identifying ANKRD55 as a novel member of the intraflagellar transport machinery. By offering significant improvements to the accuracy and coverage of human protein complexes, hu.MAP ( http://proteincomplexes.org ) serves as a valuable resource for better understanding the core cellular functions of human proteins and helping to determine mechanistic foundations of human disease.     

Proteomic approaches to uncovering virus–host protein interactions during the progression of viral infection

The integration of proteomic methods to virology has facilitated a significant breadth of biological insight into mechanisms of virus replication, antiviral host responses and viral subversion of host defenses. Throughout the course of infection, these cellular mechanisms rely heavily on the formation of temporally and spatially regulated virus–host protein–protein interactions. Reviewed here are proteomic-based approaches that have been used to characterize this dynamic virus–host interplay. Specifically discussed are the contribution of integrative mass spectrometry, antibody-based affinity purification of protein complexes, cross-linking and protein array techniques for elucidating complex networks of virus–host protein associations during infection with a diverse range of RNA and DNA viruses. The benefits and limitations of applying proteomic methods to virology are explored, and the contribution of these approaches to important biological discoveries and to inspiring new tractable avenues for the design of antiviral therapeutics is highlighted.     

生物质谱技术在蛋白质组学研究中的应用

随着技术的进步,蛋白质组学的研究重心由最初旨在鉴定细胞或组织内基因组所表达的全部蛋白质转移到从整个蛋白质组水平上阐述包括蛋白翻译后修饰、生物大分子相互作用等反映蛋白质功能的层次。多种质谱离子化技术的突破使质谱技术成为蛋白质组学研究必不可少的手段。质谱技术联合蛋白质组学多角度、深层次探索生命系统分子本质成为现阶段生命科学研究领域的主旋律之一。本文简要综述了肽和蛋白质等生物大分子质谱分析的原理、方式和应用,并对其发展前景做出展望。     

Optimization of Data-Dependent Acquisition Parameters for Coupling High-Speed Separations with LC-MS/MS for Protein Identifications

Recent developments in chromatography, such as ultra-HPLC and superficially porous particles, offer significantly improved peptide separation. The narrow peak widths, often only several seconds, can permit a 15-min liquid chromatography run to have a similar peak capacity as a 60-min run using traditional HPLC approaches. In theory, these larger peak capacities should provide higher protein coverage and/or more protein identifications when incorporated into a proteomic workflow. We initially observed a decrease in protein coverage when implementing these faster chromatographic approaches, due to data-dependent acquisition (DDA) settings that were not properly set to match the narrow peak widths resulting from newly implemented, fast separation techniques. Oversampling of high-intensity peptides lead to low protein-sequence coverage, and tandem mass spectra (MS/MS) from lower-intensity peptides were of poor quality, as automated MS/MS events were occurring late on chromatographic peaks. These observations led us to optimize DDA settings to use these fast separations. Optimized DDA settings were applied to the analysis of Trypanosome brucei peptides, yielding peptide identifications at a rate almost five times faster than previously used methodologies. The described approach significantly improves protein identification workflows that use typical available instrumentation.     

The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.     

Definitive screening design enables optimization of LC–ESI–MS/MS parameters in proteomics

In proteomics, more than 100,000 peptides are generated from the digestion of human cell lysates. Proteome samples have a broad dynamic range in protein abundance; therefore, it is critical to optimize various parameters of LC–ESI–MS/MS to comprehensively identify these peptides. However, there are many parameters for LC–ESI–MS/MS analysis. In this study, we applied definitive screening design to simultaneously optimize 14 parameters in the operation of monolithic capillary LC–ESI–MS/MS to increase the number of identified proteins and/or the average peak area of MS1. The simultaneous optimization enabled the determination of two-factor interactions between LC and MS. Finally, we found two parameter sets of monolithic capillary LC–ESI–MS/MS that increased the number of identified proteins by 8.1% or the average peak area of MS1 by 67%. The definitive screening design would be highly useful for high-throughput analysis of the best parameter set in LC–ESI–MS/MS systems.     

Mass spectrometry is only one piece of the puzzle in clinical proteomics.

Biomarker discovery in clinical proteomics is being performed on relatively large patient cohorts by utilizing the high throughput of laser desorption/ionization mass spectrometry (MALDI- and SELDI-TOF-MS). Dealing directly with patient samples as opposed to working in cell or animal systems requires a host of considerations both before and after mass spectrometric analysis to obtain robust biomarker candidates. The challenges associated with the heterogeneity of typical samples are amplified by the ability to detect hundreds to thousands of proteins simultaneously. Adherence to protocols and consistency, however, can ensure optimal results. A study starts necessarily with a relevant clinical question and proceeds to a planning phase where sample availability, statistical test selection, logistics and bias reduction are key points. The physical analysis requires consistency and standardized protocols that are helped significantly through automation. Data analysis is broken into two stages, screening and final testing, which can detect either single candidates or a pattern of proteins. Biomarker identification can be performed at this point and will help significantly in the last stage, interpretation. Replication should be performed in an independent sample set in a separate study. The candidate biomarkers from an initial study give a wealth of information that can help to pinpoint patient subpopulations for a more exhaustive proteomic study using complementary platforms with limited capacity but extremely high information content. A clinical proteomics pilot project can also lead to better selection of model systems by providing a direct link with patient samples.     

An integrated workflow for charting the human interaction proteome: insights into the PP2A system

Protein complexes represent major functional units for the execution of biological processes. Systematic affinity purification coupled with mass spectrometry (AP‐MS) yielded a wealth of information on the compendium of protein complexes expressed in Saccharomyces cerevisiae. However, global AP‐MS analysis of human protein complexes is hampered by the low throughput, sensitivity and data robustness of existing procedures, which limit its application for systems biology research. Here, we address these limitations by a novel integrated method, which we applied and benchmarked for the human protein phosphatase 2A system. We identified a total of 197 protein interactions with high reproducibility, showing the coexistence of distinct classes of phosphatase complexes that are linked to proteins implicated in mitosis, cell signalling, DNA damage control and more. These results show that the presented analytical process will substantially advance throughput and reproducibility in future systematic AP‐MS studies on human protein complexes.