DIP, the Database of Interacting Proteins: a research tool for studying cellular networks of protein interactions

The Database of Interacting Proteins (DIP: http://dip.doe-mbi.ucla.edu) is a database that documents experimentally determined protein–protein interactions. It provides the scientific community with an integrated set of tools for browsing and extracting information about protein interaction networks. As of September 2001, the DIP catalogs ~11 000 unique interactions among 5900 proteins from >80 organisms; the vast majority from yeast, Helicobacter pylori and human. Tools have been developed that allow users to analyze, visualize and integrate their own experimental data with the information about protein–protein interactions available in the DIP database.     

The MiSink Plugin: Cytoscape as a graphical interface to the Database of Interacting Proteins

The MiSink Plugin converts Cytoscape, an open-source bioinformatics platform for network visualization, to a graphical interface for the database of interacting proteins (DIP: http://dip.doe-mbi.ucla.edu). Seamless integration is possible by providing bi-directional communication between Cytoscape and any Web site supplying data in XML or tab-delimited format. Availability: MiSink is freely available for download at http://dip.doe-mbi.ucla.edu/Software.cgi.     

Coevolution of Interacting Fertilization Proteins

Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female–male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive speciation.     

The Molecular Biology Database Collection: 2003 update

The Molecular Biology Database Collection is an online resource listing key databases of value to the biological community. This Collection is intended to bring fellow scientists' attention to high-quality databases that are available throughout the world, rather than just be a lengthy listing of all available databases. As such, this up-to-date listing is intended to serve as the jumping-off point from which to find specialized databases that may be of use in advancing biological research. The databases included in this Collection provide new value to the underlying data by virtue of curation, new data connections or other innovative approaches. Short, searchable summaries and updates for each of the databases included in this Collection are available through the Nucleic Acids Research Web site at http://nar.oupjournals.org.     

The Molecular Biology Database Collection: 2002 update

The Molecular Biology Database Collection is an online resource listing key databases of value to the biological community. This Collection is intended to bring fellow scientists’ attention to high-quality databases that are available throughout the world, rather than just be a lengthy listing of all available databases. As such, this up-to-date listing is intended to serve as the initial point from which to find specialized databases that may be of use in biological research. The databases included in this Collection provide new value to the underlying data by virtue of curation, new data connections or other innovative approaches. Short, searchable summaries and updates for each of the databases included in the Collection are available through the Nucleic Acids Research Web site at http://nar.oupjournals.org.     

The RNA Modification Database: 1999 update.

The RNA Modification Database (http://medlib.med.utah.edu/RNAmods/) provides a comprehensive listing of naturally modified nucleosides in RNA. Each file includes: chemical structure; common name and symbol; type(s) of RNA in which found and corresponding phylogenetic distribution; Chemical s registry number and index name; and initial literature citations for structure characterization and chemical synthesis. New features include capability to search database files by name or substructural features, modifications in tmRNA, and links to related data and sites.     

The Database of Ribosomal Cross-links: an update.

The Database of Ribosomal Cross-links (DRC) was created in 1997. Here we describe new data incorporated into this database and several new features of the DRC. The DRC is freely available via World Wide Web at http://visitweb.com/database/ or http://www. mpimg-berlin-dahlem.mpg.de/ approximately ag_ribo/ag_brimacombe/drc/     

Identification of Connexin-43 Interacting Proteins

Connexin-43 (Cx43), the most ubiquitously expressed vertebrate gap junction protein, has been shown to interact directly with Zonula Occludens-1 (ZO-1). Although several potential functions have been proposed for the ZO-1/Cx43 interaction, the role that ZO-1 and other Cx43-interacting partners play in the regulation of Cx43 trafficking, assembly, gating and turnover are not well understood. We believed a thorough analysis and classification of other Cx43-interacting proteins might help us to understand and better test these roles. We approached this question by utilizing Tandem Mass Spectrometry (MS/MS) analysis to identify proteins from normal rat kidney whole cell lysates that could interact with the C-terminal region of Cx43. Comparison against protein sequence databases identified 19 probable protein matches, including kinases, phosphatases, membrane receptors, cell signaling molecules and scaffolding proteins. We have further characterized some of these interacting proteins, including Zonula Occludens-2 (ZO-2), via western blotting and “pull down” experiments. Further in vitro/in vivoanalysis of these interacting proteins will help in our understanding of the global role of connexins in regulating development, cell metabolism and growth.     

截断型线粒体凋亡诱导因子相互作用蛋白质的筛选与鉴定

线粒体凋亡诱导因子(AIF)是一类存在于线粒体内外膜之间保守的黄素蛋白,它从线粒体到细胞核的转位可诱导细胞凋亡．AIF诱导细胞凋亡的确切机制还不是很清楚．为了筛选可能与截断型AIF存在相互作用的蛋白质,探讨截断型AIF诱导细胞凋亡的机制,用逆转录PCR扩增剪切掉线粒体定位信号的人AIF基因片段并克隆入pcDNA3.1载体,导入肝癌HepG2细胞．成功检测到截断型在转录与翻译水平上的表达．提取转染截断型AIF基因的细胞的蛋白质,用免疫沉淀方法分离与截断型可能存在相互作用的蛋白质,用基质辅助激光解析电离飞行时间质谱对分离得到的蛋白质进行鉴定,其中一蛋白质条带经鉴定为β-actin．再次用反向免疫共沉淀法与Western blot法对筛选出的可能相互作用蛋白质进行验证．结果表明,截断型AIF与β-actin之间可能存在相互作用．     

Human Cytidine Triphosphate Synthetase 1 Interacting Proteins

We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.     

mPem蛋白相作用分子的筛选与鉴定

mPem是同源异型框基因,其C末端编码同源异型域。为进一步研究mPem蛋白在胚胎发育和生殖组织发育过程中的作用,利用GAL4酵母双杂交系统筛选小鼠7d胚胎cDNA文库,共获得3个与mPem蛋白相作用的分子。其中之一为Mdfic,一种新的转录调控因子。进一步的酵母双杂交和体外GST_Pulldown试验再次确认两者之间的相互作用。Mdfic与mPem蛋白之间的相互作用暗示两者有可能组成转录调控复合体,共同参与胚胎分化的调控,为两种蛋白功能的研究提供新的思路。     

Revealing of Proteins Interacting with Na,K-ATPase

Proteins interacting with 11-type of Na,K-ATPase were revealed in pig kidney outer medulla and duck salt glands using three different methods (immunoprecipitation, protein overlay, and chemical cross-linking). Immunoprecipitation was performed after solubilization of protein homogenate with Triton X-100 so that both membrane and cytosol proteins bound to Na,K-ATPase could be revealed. Two other methods were used to study the interaction of cytosol proteins with purified Na,K-ATPase. The sets of proteins revealed by each method in outer medulla of pig kidney were different. Proteins interacting with Na,K-ATPase that have molecular masses 10, 15, 70, 75, 105, 120, and 190 kD were found using the immunoprecipitation method. The chemical cross-linking method revealed proteins with molecular masses 25, 35, 40, 58, 68-70, and 86-88 kD. The protein overlay method revealed in the same tissue proteins with molecular masses 38, 42, 43, 60, 62, 66, 70, and 94 kD.     

神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白

近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用.     

HeLa细胞中与组织型转谷氨酰胺酶相互作用蛋白质的筛选

组织型转谷氨酰胺酶 (tissuetransglutaminase ,tTG ,TGII)是转谷氨酰胺酶家族成员之一 ,多数细胞凋亡过程中均有tTG表达水平的升高。为研究tTG在细胞凋亡过程中发挥作用的机制 ,利用Gal 4酵母双杂交系统筛选了HeLa细胞中与tTG相互作用的蛋白质 ,获得了 17个阳性酵母克隆。序列测定显示其中 1个克隆所含cDNA序列编码TIA 1相关蛋白 (TIA 1 relatedprotein ,TIAR )C端 12 9个氨基酸残基序列 ,GST下拉 (pull down)实验也证实tTG与TIAR能相互作用 ,而且这种相互作用需要Ca2 参与作用。这些结果提示tTG可能通过其Ca2 依赖的转谷氨酰胺活性对TIAR进行修饰从而影响TIAR的功能 ,可能在细胞凋亡中发挥着一定的作用。     

Yeast two-hybrid方法筛选VISA相互作用蛋白

病原微生物感染对人类健康构成巨大威胁,先天免疫系统是生物体在长期进化过程中建立起来的天然保护系统。VISA(MAVS,CARDIF,IPS-1[1-3])是连接胞浆dsRNA受体RIG-I与下游信号转导通路的一个接头蛋白,研究结果表明VISA无论在TLR3非依赖的或者TLR3介导的抗病毒IFN信号途径中都具有关键作用,但目前对VISA信号转导的详细机制还不十分清楚。更多VISA相互作用蛋白的发现以及它们之间的作用机制的研究能完善我们对VISA参与的信号转导机制的认识。利用酵母双杂交系统,用VISA蛋白的全长做诱饵(Bait)筛选293 T细胞c DNA表达文库,并结合免疫共沉淀实验,双荧光素酶报告基因系统验证筛选到的阳性克隆和VISA作用的真实性。通过酵母双杂交系统成功筛选到Sec13L1候选基因并验证了其与VISA的全长蛋白相互作用,在293 T细胞中过表达该基因,研究结果显示Sec13L1过表达能促进VISA对IFNβ的诱导,并存在剂量效应。     

植物中验证蛋白相互作用的Pull-down和Co-IP技术

蛋白互作在细胞生命活动中发挥关键作用,在不同时空层面上参与多种细胞学过程,因此研究蛋白互作对理解分子调控网络至关重要。通常情况下,利用酵母双杂交系统筛选植物蛋白互作必须通过体外和体内系统进行验证。Pull-down和Co-IP是验证植物蛋白互作的常用技术。Pull-down被广泛用于体外验证蛋白间的直接互作;而在植物活体内,利用本氏烟草(Nicotiana benthamiana)叶片瞬时表达蛋白,继而通过Co-IP进行鉴定是目前验证蛋白互作最简单且最有效的方法之一。该文对GST Pull-down和烟草瞬时表达系统中Co-IP技术原理及实验方案进行详细描述,以期为验证植物蛋白互作提供参考。     

The Evolution of the MAP Kinase Pathways: Coduplication of Interacting Proteins Leads to New Signaling Cascades

The MAP-kinase pathways are intracellular signaling modules that are likely to exist in all eukaryotes. We provide an evolutionary model for these signaling pathways by focusing on the gene duplications that have occurred since the divergence of animals from yeast. Construction of evolutionary trees with confidence assessed by bootstrap clearly shows that the mammalian JNK and p38 pathways arose from an ancestral hyperosmolarity pathway after the split from yeast and before the split from C. elegans. These coduplications of interacting proteins at the MAPK and MEK levels have since evolved toward substrate specificity, thus giving distinct pathways. Mammalian duplications since the split from C. elegans are often associated with divergent tissue distribution but do not appear to confer detectable substrate specificity. The yeast kinase cascades have undergone similar fundamental functional changes since the split from mammals, with duplications giving rise to central signaling components of the filamentous and hypoosmolarity pathways. Experimentally defined cross-talk between yeast pheromone and hyperosmolarity pathways is mirrored with corresponding cross-talk in mammalian pathways, suggesting the existence of ancient orthologous cross-talk; our analysis of gene duplications at all levels of the cascade is consistent with this model but does not always provide significant bootstrap support. Our data also provide insights at different levels of the cascade where conflicting experimental evidence exists. Received: 2 December 1998 / Accepted: 9 June 1999     

Proteomics Uncovers Proteins Interacting Electrostatically with Thioredoxin in Chloroplasts

The ability of thioredoxin f to form an electrostatic (non-covalent) complex, earlier found with fructose-1,6-bisphosphatase, was extended to include 27 previously unrecognized proteins functional in 11 processes of chloroplasts. The proteins were identified by combining thioredoxin f affinity chromatography with proteomic analysis using tandem mass spectrometry. The results provide evidence that an association with thioredoxin enables the interacting protein to achieve an optimal conformation, so as to facilitate: (i) the transfer of reducing equivalents from the ferredoxin/ferredoxin-thioredoxin reductase complex to a target protein; (ii) in some cases, to enable the channeling of metabolite substrates; (iii) to function as a subunit in the formation of multienzyme complexes.     

转录因子X-box结合蛋白1相互作用蛋白的筛选和鉴定

X-box 结合蛋白 1 是一种重要的转录因子,参与体内多项信号转导过程. 为进一步研究 XBP1 的生物学功能,运用酵母双杂交技术在肝细胞文库中筛选 XBP1 的结合蛋白. 首先运用 PCR 技术扩增获得 XBP1 的编码序列,克隆至 pGEM-T 载体,经测序鉴定后,亚克隆至诱饵载体 pGBKT7 中,转化酵母 AH109(a type). 免疫印迹检测诱饵质粒 pGBKT7-XBP1 在AH109 酵母中的表达之后,含有诱饵质粒的酵母 AH109 与含有肝细胞 cDNA 文库质粒 pACT2 的酵母 Y187(αtype)配合,配合后的二倍体酵母生长在含有 X-α-gal 的营养缺陷型培养基上 (SD/-Trp-Leu-His-Ade) 进行选择和筛选,经测序和序列比对确定阳性克隆的开放读码框 ORF,得到 7 种不同的蛋白质. 为了进一步验证这些筛选蛋白质与 XBP1 的相互作用,克隆其中一种蛋白质 MT1E,并运用 GST pulldown 和免疫共沉淀技术成功检测了 MT1E 和 XBP1 的相互作用(体外 / 体内),结果提示,MT1E 可能是 XBP1 的一个新的调节蛋白. 通过酵母双杂交技术筛选得到的 7 种蛋白质分别与肝细胞基础代谢、蛋白质的合成与运输、细胞的增殖与凋亡密切相关. 上述结果有助于揭示 XBP1 的生物学功能,为进一步探讨 XBP1 的表达和调控机制提供新线索.