Combining different 'omics' technologies to map and validate protein-protein interactions in humans.

The mapping of protein-protein interactions is key to understanding biological processes. Many technologies have been reported to map interactions and these have been systematically applied in yeast. To date, the number of reported yeast protein interactions that have been truly validated by at least one other approach is low. The mapping of human protein interaction networks is even more complicated. Thus, it is unreasonable to try to map the human interactome; instead, interaction mapping in human cell lines should be focused along the lines of diseases or changes that can be associated with specific cells. In this paper, an approach for combining different 'omics' technologies to achieve efficient mapping and validation of protein interactions in human cell lines is presented.     

大规模的酵母双杂交系统在蛋白质相互作用组图谱中的应用

蛋白质-蛋白质之间的相互作用是蛋白质发挥其功能的重要途径之一。通过研究蛋白质组中所有蛋白质之间的相互作用做出蛋白质相互作用对图谱是功能基因组时代许多科学家关注的问题,而大规模的酵母双杂交系统是蛋白质相互作用对图谱的研究中应用较为广泛的策略。近两年来该策略最具代表的实例是用它进行酵母中所有蛋白之间相互作用的检查。但是巨大的蛋白质网络比我们想象要大得多,单一的双杂交系统不能解决所有问题,需要同其它的方法有效地结合。     

Detection of protein-protein interactions using nonimmune IgG and BirA-mediated biotinylation

Detection of protein-protein interactions in cells is crucial for understanding the biological functions of proteins, including their roles in signal transduction. However, current methods require specific antibodies both for immunoprecipitation and detection, making them expensive and sometimes unreliable. Here we describe protocols for protein-protein interaction assays that use nonimmune IgG-conjugated Sepharose to precipitate the IgG binding domain (ZZ) fused to the bait protein; the interaction partner is fused to Avitag and biotinylated by BirA so that it can be detected by a one-step blot with Dylight 680 streptavidin to detect the Avitag fusion protein. Since this method does not require specific antibodies and is inexpensive, sensitive, and reliable, it should be useful for detecting protein-protein interactions in cells.     

Molecular dynamics shows complex interplay and long-range effects of post-translational modifications in yeast protein interactions

Post-translational modifications (PTMs) play a vital, yet often overlooked role in the living cells through modulation of protein properties, such as localization and affinity towards their interactors, thereby enabling quick adaptation to changing environmental conditions. We have previously benchmarked a computational framework for the prediction of PTMs’ effects on the stability of protein-protein interactions, which has molecular dynamics simulations followed by free energy calculations at its core. In the present work, we apply this framework to publicly available data on Saccharomyces cerevisiae protein structures and PTM sites, identified in both normal and stress conditions. We predict proteome-wide effects of acetylations and phosphorylations on protein-protein interactions and find that acetylations more frequently have locally stabilizing roles in protein interactions, while the opposite is true for phosphorylations. However, the overall impact of PTMs on protein-protein interactions is more complex than a simple sum of local changes caused by the introduction of PTMs and adds to our understanding of PTM cross-talk. We further use the obtained data to calculate the conformational changes brought about by PTMs. Finally, conservation of the analyzed PTM residues in orthologues shows that some predictions for yeast proteins will be mirrored to other organisms, including human. This work, therefore, contributes to our overall understanding of the modulation of the cellular protein interaction networks in yeast and beyond.     

Unraveling regulation and new components of human P-bodies through a protein interaction framework and experimental validation

The cellular factors involved in mRNA degradation and translation repression can aggregate into cytoplasmic domains known as GW bodies or mRNA processing bodies (P-bodies). However, current understanding of P-bodies, especially the regulatory aspect, remains relatively fragmentary. To provide a framework for studying the mechanisms and regulation of P-body formation, maintenance, and disassembly, we compiled a list of P-body proteins found in various species and further grouped both reported and predicted human P-body proteins according to their functions. By analyzing protein-protein interactions of human P-body components, we found that many P-body proteins form complex interaction networks with each other and with other cellular proteins that are not recognized as P-body components. The observation suggests that these other cellular proteins may play important roles in regulating P-body dynamics and functions. We further used siRNA-mediated gene knockdown and immunofluorescence microscopy to demonstrate the validity of our in silico analyses. Our combined approach identifies new P-body components and suggests that protein ubiquitination and protein phosphorylation involving 14-3-3 proteins may play critical roles for post-translational modifications of P-body components in regulating P-body dynamics. Our analyses provide not only a global view of human P-body components and their physical interactions but also a wealth of hypotheses to help guide future research on the regulation and function of human P-bodies.     

蛋白质相互作用数据库及其应用

对蛋白质相互作用及其网络的了解不仅有助于深入理解生命活动的本质和疾病发生的机制,而且可以为药物研发提供靶点.目前,通过高通量筛选、计算方法预测和文献挖掘等方法,获得了大批量的蛋白质相互作用数据,并由此构建了很多内容丰富并日益更新的蛋白质相互作用数据库.本文首先简要阐述了大规模蛋白质相互作用数据产生的3种方法,然后重点介绍了几个人类相关的蛋白质相互作用公共数据库,包括HPRD、BIND、 IntAct、MINT、 DIP 和MIPS,并概述了蛋白质相互作用数据库的整合情况以及这些数据库在蛋白质相互作用网络构建上的应用.     

Convergent evolution with combinatorial peptides

Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.     

Pairwise interactions of the six human MCM protein subunits

The eukaryotic minichromosome maintenance (MCM) proteins have six subunits, Mcm2 to 7p. Together they play essential roles in the initiation and elongation of DNA replication, and the human MCM proteins present attractive targets for potential anticancer drugs. The six MCM subunits interact and form a ring-shaped heterohexameric complex containing one of each subunit in a variety of eukaryotes, and subcomplexes have also been observed. However, the architecture of the human MCM heterohexameric complex is still unknown. We systematically studied pairwise interactions of individual human MCM subunits by using the yeast two-hybrid system and in vivo protein-protein crosslinking with a non-cleavable crosslinker in human cells followed by co-immunoprecipitation. In the yeast two-hybrid assays, we revealed multiple binary interactions among the six human MCM proteins, and a subset of these interactions was also detected as direct interactions in human cells. Based on our results, we propose a model for the architecture of the human MCM protein heterohexameric complex. We also propose models for the structures of subcomplexes. Thus, this study may serve as a foundation for understanding the overall architecture and function of eukaryotic MCM protein complexes and as clues for developing anticancer drugs targeted to the human MCM proteins.     

Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system

The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.     

PRINCESS, a protein interaction confidence evaluation system with multiple data sources

Advances in proteomics technologies have enabled novel protein interactions to be detected at high speed, but they come at the expense of relatively low quality. Therefore, a crucial step in utilizing the high throughput protein interaction data is evaluating their confidence and then separating the subsets of reliable interactions from the background noise for further analyses. Using Bayesian network approaches, we combine multiple heterogeneous biological evidences, including model organism protein-protein interaction, interaction domain, functional annotation, gene expression, genome context, and network topology structure, to assign reliability to the human protein-protein interactions identified by high throughput experiments. This method shows high sensitivity and specificity to predict true interactions from the human high throughput protein-protein interaction data sets. This method has been developed into an on-line confidence scoring system specifically for the human high throughput protein-protein interactions. Users may submit their protein-protein interaction data on line, and the detailed information about the supporting evidence for query interactions together with the confidence scores will be returned. The Web interface of PRINCESS (protein interaction confidence evaluation system with multiple data sources) is available at the website of China Human Proteome Organisation.     

Protein-protein complexation in bioluminescence

In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an “accessory protein” whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an “antenna protein” altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca²⁺-regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of X-ray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.     

Interactive proteomics research technologies: recent applications and advances

Proteins rarely exert their function alone. They normally function in multiprotein complexes that play central roles in all biological functions. Thus, it is not surprising that the investigation of protein-protein interactions on a global scale, of so-called interactomes, has become crucial to modern molecular biology. Dissecting partners in protein complexes gives insight into their molecular function and can help in understanding disease-related mechanisms, ultimately resulting in better drug target definition. A variety of methods exist to unravel protein interaction circuitries and recently, significant progress has been made in adapting these tools for the generation of large-scale interaction datasets. Here, we present an overview of the latest advances and applications of interactive proteomics research technologies.     

Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening

Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.     

SNAVI: Desktop application for analysis and visualization of large-scale signaling networks

Background  

Studies of cellular signaling indicate that signal transduction pathways combine to form large networks of interactions. Viewing protein-protein and ligand-protein interactions as graphs (networks), where biomolecules are represented as nodes and their interactions are represented as links, is a promising approach for integrating experimental results from different sources to achieve a systematic understanding of the molecular mechanisms driving cell phenotype. The emergence of large-scale signaling networks provides an opportunity for topological statistical analysis while visualization of such networks represents a challenge.     

Application of hydrogen/deuterium exchange mass spectrometry to study protein tyrosine phosphatase dynamics, ligand binding, and substrate specificity

Protein tyrosine phosphatases (PTPs) are signaling enzymes that control a diverse array of cellular processes. Further insight into the specific functional roles of PTPs in cellular signaling requires detailed understanding of the molecular basis for substrate recognition by the PTPs. A central question is how PTPs discriminate between multiple structurally diverse substrates that they encounter in the cell. Although X-ray crystallography is capable of revealing the intimate structural details for molecular interaction, structures of higher order PTP.substrate complexes are often difficult to obtain. Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) is a powerful tool for mapping protein-protein interfaces, as well as identifying conformational and dynamic perturbations in proteins. In addition, H/DX-MS enables analysis of large protein complexes at physiological concentrations and provides insight into the solution behavior of these complexes that can not be gleaned from crystal structures. We have utilized H/DX-MS to probe PTP dynamics, ligand binding, and the structural basis of substrate recognition. In this article, we review general principles of H/DX-MS technology as applied to study protein-protein interactions and dynamics. We also provide protocols for H/DX-MS successfully used in our laboratory to define the molecular basis of ERK2 substrate recognition by MKP3. Many of the aspects that we cover in detail should be applicable to the study of other PTPs with their specific targets.     

Contemporary strategies for the stabilization of peptides in the alpha-helical conformation

Herein we review contemporary synthetic and protein design strategies to stabilize the alpha-helical motif in short peptides and miniature proteins. Advances in organometallic catalyst design, specifically for the olefin metathesis reaction, enable the use of hydrocarbon bridges to either crosslink side chains of specific residues or mimic intramolecular hydrogen bonds with carbon-carbon bonds. The resulting hydrocarbon-stapled and hydrogen bond surrogate alpha-helices provide unique synthetic ligands for targeting biomolecules. In the protein design realm, several classes of miniature proteins that display stable helical domains have been engineered and manipulated with powerful in vitro selection technologies to yield libraries of sequences that retain their helical folds. Rational re-design of these scaffolds provide distinctive reagents for the modulation of protein-protein interactions.